Munich - (DGI) und des Deutschen Zentrums für Infektionsforschung

Munich
W el c o m e N o te o f th e C o nf erenc e C h ai rs
Dear C olleagues,
We are delighted to invite you to the J oint DGI and DZIF Annual Meeting 2015 in Munich.
Infectious diseases are a global challenge – evidenced by the ongoing outbreak of Ebola, the Influenza wave
striking us, and the increase in bacterial multi-drug resistance, significantly impacting human health.
We believe that these challenges can only be met by a j oint effort of basic scientists and clinicians in infection
medicine. The German Society of Infectious Diseases (DGI) and the German C enter for Infection Research (DZIF)
have therefore set up a j oint conference programme addressing current topics in the field of infectious disease
research and patient care.
The agenda will cover recent developments from all Thematic Translational Units of the DZIF, highlight novel
advances in diagnostics and treatment, and offer symposia for clinical education in infectious diseases.
The programme is designed to enforce exchange between researchers and clinicians, and will provide ample
opportunities for networking between different disciplines in infection medicine.
We are looking forward to meeting you in Munich on 19 – 21 November, 2015!
Liebe Kolleginnen, liebe Kollegen,
hiermit laden wir Sie herzlich zur diesj ä hrigen Gemeinsamen J ahrestagung der DGI und des DZIF in München
ein.
Infektionskrankheiten stellen eine weltweite medizinische Herausforderung dar – Beispiele wie der aktuelle
Ebola-Ausbruch, die j ä hrliche Grippe-Welle oder die globale Resistenzentwicklung bakterieller Krankheitserreger verdeutlichen dies. Die Deutsche Gesellscha für Infektiologie (DGI) und das Deutsche Zentrum für Infektionsforschung (DZIF) haben ein gemeinsames Konferenzprogramm erstellt, welches sich an Grundlagenwissensc a ler und klinisch tä tige Kollegen im Bereich der Infektionsmedizin richtet. Neben neuen wissensc a lichen
Entwicklungen in der iagnostik und Therapie von Infektionskrank eiten aus allen TTU-Bereichen des DZIF
werden klinisch orientierte Symposien und Workshops angeboten. Wir mö chten auf diesem Weg den intensiven Austausch zwischen Wissensc a lern und Klinikern fö rdern und die Vernetzung unterschiedlicher Disziplinen in der Infektions edi in unterstützen.
Wir freuen uns sehr, Sie vom 19 .– 21. November 2015 in München begrüßen zu dürfen!
U l ri k e Pro tz er
D i rk B u sc h
For the German C enter for Infection Research (DZIF)
S u sanne H ero l d
G erd F ä tk enh eu er
For the German Society of Infectious Diseases (DGI)
Organisation and Imprint
Venue
Paulaner am Nockherberg
Hochstrasse 77
81541 Munich
Date
19–21 November 2015
Conference Website
www.dgi-dzif-kongress2015.de
Organiser
Conventus on behalf of the German Society of Infectious Diseases (DGI) and the German Center for Infection
Research (DZIF)
www.dgi-net.de • www.dzif.de
Conference Chairs
Prof. Dr. med. Ulrike Protzer
Technical University of Munich / Helmholtz Zentrum Munich
Institute of Virology
Trogerstrasse 30
81675 Munich
Prof. Dr. med. Dirk Busch
Technical University of Munich
Institute for Medical Microbiology, Immunology and Hygiene
Trogerstrasse 30
81675 Munich
Prof. Dr. med. Susanne Herold
Justus-Liebig-University Giessen
Department of Internal Medicine II
Klinikstrasse 33
35392 Giessen
Prof. Dr. med. Gerd Fätkenheuer
University Hospital Cologne
Department of Internal Medicine
Kerpener Strasse 62
50937 Cologne
Professional Congress Organiser
Conventus Congressmanagement & Marketing GmbH
Roxelane Görls
Carl-Pulfrich-Strasse 1
07745 Jena
Tel. +49 3641 31 16-312
Fax +49 3641 31 16-243
[email protected]
www.conventus.de
Design/Layout
Layout
Print
Circulation
Editorial Deadline
www.krea.tif-design.de
www.kerndruck.de
400
9 November, 2015
Table of Content Scientific Programme – Overview ............................................................................................. VI Scientific Programme – Thursday, 19 November 2015 ............................................................ VII Scientific Programme – Friday, 20 November 2015 .................................................................. IX Scientific Programme – Saturday, 21 November 2015 ............................................................. XI Sponsors .................................................................................................................................. XIII Industrial Exhibition ................................................................................................................. XIV SPEED TALKS ............................................................................................................................... 1 Symposium I – Emerging Infections and Pneumonia .......................................................................... 1 Symposium II – Infections of the immunocompromised Host ............................................................ 6 Symposium III – Mycobacteria, Tuberculosis and Malaria ................................................................ 12 Symposium IV – Antibiotic resistant bacterial infections .................................................................. 18 Symposium V – Chronic Viral Infections ........................................................................................... 24 POSTER ..................................................................................................................................... 30 Emerging Infections and Pneumonia ................................................................................................ 30 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections .......... 62 Mycobacteria, Tuberculosis and Malaria ........................................................................................ 102 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship ........... 118 Chronic Viral Infections – HIV and Hepatitis ................................................................................... 166 Index of Authors ..................................................................................................................... 212 Notes ...................................................................................................................................... XVII Social Programme .................................................................................................................. XVIII General Information .............................................................................................................. XVIII Scientific Programme – Overview Friday, 20 November 2015
Thursday, 19 November 2015
08:30–10:15
Symposium IV
Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship
Saturday, 21 November 2015
08:15–09:15
08:15–09:15
Akademie für Infektionsmedizin Akademie Kompakt "Tropen‐ und Reisemedizin" Karrieremöglich‐
keiten – Der Infektiologe
Industrieausstellung und Kaffeepause
09:30–09:50 Preisvortrag 09:50–10:30
Industrial exhibition and coffee break
11:00–11:15 Congress opening
11:15–12:30
Symposium VI
Neue Substanzen, neue Therapiestrategien 10:30–10:50 Infektionen bei Asylsuchenden 10:45–12:30
10:50–11:50
Symposium V
Chronic Viral Infections: HIV and Hepatitis
Symposium VII
Infektiologie Update ‐ Neues zum Thema…
Symposium I
Emerging Infections and Pneumonia
Industrieausstellung und Kaffeepause
12:20–13:20
Industrial exhibition and lunch break
Symposium VIII
Multiresistente und hochkontagiöse Erreger
13:10–14:00
13:20–13:30 Kongressverabschiedung
12:30–13:00
DZIF Award: Translational Infection Research
Industrial exhibition and lunch break
Poster Session II
13:45–14:30
Poster Session I
14:00–14:30
Plenary Lecture by E. Pamer
14:30–16:15
14:30–15:15
Symposium II
Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections
Infektiologie Quiz
Industrieausstellung und Kaffeepause
15:45–16:45
Podiumsdiskussion
Industrial exhibition and coffee break
16:45–18:30
Symposium III
Mycobacteria, Tuberculosis and Malaria
16:45–17:00 Posterpreis‐Verleihung
17:00–19:00
17:00–19:00
DGI Mitglieder‐
versammlung
DZIF Mitglieder‐
versammlung
18:30–19:30
Meeting of the DGI Sections
from 19:30
Ab 20:00
Social Evening "Boarischer Abend"
Nachtführung durch München
VI Scientific Programme – Thursday, 19 November 2015 11:00–11:15 11:15–12:30 Chairs 11:15 ST 1 11:22 ST 2 11:29 11:49 ST 3 11:56 ST 4 12:03 12:23 ST 5 12:30–13:00 Chair 13:00–13:45 13:45–14:30 14:30–16:15 Chairs 14:30 ST 6 14:37 Congress Opening S. Herold (Gießen), M. Krönke (Köln), U. Protzer (München) Symposium I – Emerging Infections and Pneumonia C. Drosten (Bonn), S. Hippenstiel (Berlin) Surveillance and Outbreak response management and analysis system (SORMAS) to support the control of the Ebola Virus Disease (EVD) outbreak G. Krause (Braunschweig) Safety and reactogenicity of rVSV Vaccine Expressing Zaire Ebola virus Glycoprotein: Phase I Trial in Germany C. Dahlke (Hamburg) DGI lecture Molecular imaging of the pathogen host interaction in human lungs A. Hocke (Berlin) Building an “Ebola aid‐workers´ School” in one week – Effective and rapid set‐up of the pre‐ deployment training program for >200 staff for the Ebola Intervention in West Africa, 2014. M. Gertler (Berlin) Randomized phase I/IIa clinical trial demonstrates safety and immunogenicity of an MVA‐based influenza A/H5N1 vaccine in healthy adults G. Sutter (München) DZIF lecture Emerging Infections‐ Global challenges and Clinical Implications M. Addo (Hamburg) Murine bone marrow‐derived mesenchymal stem cells exert anti‐viral and barrier‐protective properties in influenza virus infection L. Jankauskaite (Gießen) Plenary Lecture: DZIF Award “Translational Infection Research” D. Heinz (Braunschweig) Targeting immune evasion for vaccine development against chronic infection with Helicobacter pylori M. Gerhard (München) Industrial exhibition and lunch break Poster Session I at Foyer 1 and Foyer 2 Symposium II – Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections S. Lemmen (Aachen), T. Schulz (Hannover), S. Suerbaum (Hannover) Chlorhexidine containing iv‐catheter securement dressings for the prevention of central venous catheter‐related bloodstream infections in neutropenic patients: a randomized trial (COAT study) M. Vehreschild (Köln) DZIF lecture Inhibitors of EBV oncoprotein LMP1:TRAF interaction A. Kieser (München) VII Scientific Programme – Thursday, 19 November 2015 14:57 ST 7 15:04 ST 8 15:11 15:31 ST 9 15:38 ST 10 15:45 16:05 ST 11 16:15–16:45 16:45–18:30 Chairs 16:45 ST 12 16:52 17:12 ST 13 17:19 ST 14 17:26 17:46 ST 15 17:53 ST 16 Intestinal sugars modulate microbiota composition and the susceptibility to enteric pathogens G. Grassl (Hannover) Identification of a novel compound inhibiting the type III secretion system 1 of Salmonella Typhimurium S. Wagner (Tübingen) DGI lecture Infection control strategies in patient with C. difficile A. Widmer (Basel, Switzerland) Induction of pro‐ and anti‐inflammatory human T cell funtionalities by distinct microbes C. Zielinski (München) Identification of T cell epitopes for a therapeutic HPV16 vaccine A. Riemer (Heidelberg) DZIF lecture Helicobacter pylori infection 2015 – A global challenge to human health S. Suerbaum (Hannover) Identification of novel mutations in Common Variable Immunodeficiency Syndrome (CVID) through targeted re‐sequencing of a CVID cohort F. Atschekzei (Hannover) Industrial exhibition and coffee break Symposium III – Mycobacteria, Tuberculosis and Malaria P. Hartmann (Köln), P. G. Kremsner (Tübingen), S. Niemann (Borstel) The role of filarial infections and filarial‐induced immunomodulation in the pathogenesis of epilepsy in sub‐Saharan Africa T. Wagner (Heidelberg) DZIF lecture Tracing evolution and spread of MDR M. tuberculosis using genome analysis M. Merker (Borstel) Long‐term safety and efficacy results of the PanACEA MAMS‐TB randomised controlled trial N. Heinrich (München) Characteristics of patients with multidrug‐resistant tuberculosis at the DZIF Clinical Tuberculosis Center (ClinTB), Medical Clinic Borstel I. D. Olaru (Borstel) DGI lecture Exploiting a novel host cell‐based screen for antituberculous drug discovery J. Rybniker (Köln) Host transcriptomics revealing a key to the success story of Mycobacterium tuberculosis U. von Both (München) The improved and controlled investigation of febrile illness in a rural setting in South‐Western Tanzania supports the Malaria diagnostic B. Flach (München) VIII Scientific Programme – Thursday, 19 November 2015 18:00 18:20 ST 17 18:30–19:30 From 19:30 DZIF lecture Immune response to vaccination with whole Plasmodium falciparum sporozoites B. Mordmüller (Tübingen) A randomized, controlled, double‐blind, phase 1 clinical trial to evaluate safety, tolerability, immunogenicity and efficacy of CAF01 and aluminum hydroxide as adjuvants for the malaria vaccine candidate GMZ2 in healthy adult African volunteers A. A. Adegnika (Tübingen/Lambarene, Gabun) Meeting of the DGI Sections at “Kastanienstube” Social Evening at “Raum Bayern” Scientific Programme – Friday, 20 November 2015 08:30–10:15 Chairs 08:30 ST 18 08:37 08:57 ST 19 09:04 ST 20 09:11 09:31 ST 21 09:38 ST 22 09:45 10:05 ST 23 10:15–10:45 Symposium IV – Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship K. de With (Dresden), H.‐G. Sahl (Bonn), H. Seifert (Köln) The lectin LecB as target for anti‐infectives against chronic Pseudomonas aeruginosa infections A. Titz (Saarbrücken) DZIF lecture New approaches for decolonization of antibiotic‐resistant pathogens A. Peschel (Tübingen) Development of a virulence gene scoring system for ESBL‐producing E. coli isolates from human and animal sources J. Schmiedel (Gießen) Quality assurance in blood culture diagnostics – the Thuringian prospective population‐based registry AlertsNet F. Brunkhorst (Jena) DGI lecture An evolutionary perspective on antibiotic treatment therapy H. Schulenburg (Kiel) Molecular characterization of third generation cephalosporin‐resistant Enterobacteriaceae (3GCREB) on hospital admission – results from the first ATHOS prevalence study A. Hamprecht (Köln) Characterization and quantification of antimicrobial resistance selection pressure caused by antibiotic treatment: a sequence based approach S. Peter (Tübingen) DZIF lecture Tear down the wall – Novel antibiotics from microbial dark matter targeting cell wall biosynthesis T. Schneider (Bonn) Isolation and characterization of antibacterial cyclophanes and related analogs from cyanobacteria of the genera Nostoc and Cylindrospermum T. Niedermeyer (Tübingen) Industrial exhibition and coffee break IX Scientific Programme – Friday, 20 November 2015 10:45–12:30 Chairs 10:45 ST 24 10:52 11:12 ST 25 11:19 ST 26 11:26 11:46 ST 27 11:53 ST 28 12:00 12:20 ST 29 12:30–13:10 13:10–14:00 14:00–14:30 Chair 14:30–15:15 Moderation 15:15–15:45 Symposium V – Chronic Viral Infections: HIV and Hepatitis H.‐G. Kräusslich (Heidelberg), M. Manns (Hannover), C. Stephan (Frankfurt a. M.) Targeted cleavage of the HIV‐1 proviral genome using AAV Vector‐Mediated CRISPR M. Nickl (Heidelberg) DZIF lecture Antibody‐mediated neutralization of HIV‐1 F. Klein (Köln) High‐efficiency knockout of HIV co‐receptor CCR5 in primary T cells after mRNA transfection of the novel transcription activator‐like effector nuclease CCR5‐Uco‐TALEN B. Fehse (Hamburg) Engineering and analysis of Brec1: A Broad‐range recombinase that specifically targets the majority of HIV‐1 isolates J. Hauber (Hamburg) DZIF lecture Future Challenges in Viral Hepatitis T. von Hahn (Hannover) Differential European trends in HBV prevalence over the last decades J. Ott (Braunschweig) Suppression of HBV by RNAi restores HBV‐specific immunity and enhances the efficacy of therapeutic vaccination A. Kosinska (München) DGI lecture T follicular helper cells in HIV infection H. Streeck (Essen) Impact of HLA‐B alleles on the immune evasion of hepatitis delta virus from CTL response and epitope discovery H. Karimzadeh (München) Industrial exhibition and lunch break Poster Session II at Foyer 1 and Foyer 2 Plenary Lecture D. Busch (München) Microbiota‐mediated defense against intestinal infection E. Pamer (New York, USA) Infektiologie Quiz N. Jung (Köln) Industrieausstellung und Kaffeepause X Scientific Programme – Friday, 20 November 2015 15:45–16:45 Moderation 16:45–17:00 17:00–19:00 17:00–19:00 Ab 20:00 Podiumsdiskussion Impfschutz gegen die Seuchen von morgen ‐ wann ist es ethisch, die klinische Entwicklung von Impfstoffen gegen neuauftretende Erreger zu beschleunigen? K. Zinkant (München/Berlin) M. Addo (Hamburg) S. Becker (Marburg) K. Cichutek (Langen) J. Klein (Berlin) Posterpreis‐Verleihung Überreicht durch G. Fätkenheuer (Köln) DGI Mitgliederversammlung in Saal 1 DZIF Mitgliederversammlung in Saal 2 Nachtführung durch München Scientific Programme – Saturday, 21 November 2015 08:15–09:15 Saal 1 Vorsitz 08:15–09:15 Raum Bayern Vorsitz 08:15 08:35 08:55 09:15–09:30 09:30–09:50 Vorsitz 09:50–10:30 Vorsitz 09:50 10:10 Akademie für Infektionsmedizin „Tropen‐ und Reisemedizin“ G.‐D. Burchard (Hamburg) Karrieremöglichkeiten – Der Infektiologe C. Lehmann (Köln) Die Klinikärztin R. Draenert (München) Der niedergelassene Arzt U. Kastenbauer (München) Flüchtlingsmedizin aus Sicht eines jungen Infektiologen A. Jablonka (Hannover) Industrieausstellung und Kaffeepause Preisvortrag „Förderpreis für Klinische Infektionsforschung der DGI“ S. Herold (Gießen) Therapie der schweren Malaria mit Artesunat und Hämolyse ‐ rasche Wirkung, verzögerte Nebenwirkung T. Rolling (Hamburg) Symposium VI – Neue Substanzen, neue Therapiestrategien J. Rupp (Lübeck), N. Suttorp (Berlin) Clostridium difficile – Neue Therapieansätze O. Cornely (Köln) Klinischer Einsatz neuer Antibiotika W. Kern (Freiburg) XI Scientific Programme – Saturday, 21 November 2015 10:30–10:50 10:50–11:50 Vorsitz 10:50 11:10 11:30 11:50–12:20 12:20–13:20 Vorsitz 12:20 12:40 13:00 13:20–13:30 Infektionen bei Asylsuchenden T. Löscher (München) Symposium VII – Infektiologie Update: Neues zum Thema… H. Klinker (Würzburg), O. Witzke (Essen) … HIV K. Arasteh (Berlin) … Opportunistische Infektionen A. Ullmann (Würzburg) … Staphylokokken S. Rieg (Freiburg) Industrieausstellung und Kaffeepause Symposium VIII – Multiresistente und hochkontagiöse Erreger M. Addo (Hamburg), H. Seifert (Köln) Neue Aspekte der Diagnostik – Multiresistente Erreger H. Rohde (Hamburg) Management von Patienten mit hochkontagiösen Erkrankungen T. Grünewald (Leipzig) Wen und wann isolieren? S. Lemmen (Aachen) Kongressverabschiedung D. Busch (München), G. Fätkenheuer (Köln), S. Herold (Gießen), M. Krönke (Köln) U. Protzer (München) XII S p o nso rs
We thank the following sponsors for their support:
G o l d S p o nso r
J anssen-C ilag GmbH (Neuss)
B ro nz e S p o nso rs
Astellas P harma GmbH (Munich)
Bristol-Myers Squibb GmbH & C o. KGaA (Munich)
Gilead Sciences GmbH (Martinsried)
MSD Sharp & Dohme GmbH (Haar)
ViiV Healthcare GmbH (Munich)
S p o nso r o f N am e B adg e
RIEMSER P harma GmbH (Greifswald)
F u rth er S p o nso r
Boehringer Ingelheim GmbH (Ingelheim a. R.)
T ransp arenc y
The members of the association “ Freiwillige Selbstkontrolle für die Arznei ittelindustrie e. V. (FSA)” (Voluntary
Self-regulation for the P harmaceutical Industry) have redefined the FSA codex for claiming more transparency.
In future congress organizers are obliged to inform potential conference participants about the scope and
terms of the assistance of the pharmaceutical industry, prior to the event.
We comply with this regulation and inform you about the sponsoring amount of the participating
pharmaceutical companies: Astellas P harma GmbH 5,000 EUR; Boehringer Ingelheim GmbH 2,000 EUR; BristolMyers Squibb GmbH 5,000 EUR; Gilead Sciences GmbH 5,000 EUR; J anssen-C ilag GmbH 15,000 EUR; MSD Sharp
& Dohme GmbH 5,000 EUR; ViiV Healthcare GmbH 5,000 EUR.
X III
Industrial Exhibition The following companies look forward to meeting you onsite: Company AID Autoimmun Diagnostika GmbH (Strassberg)
Akademie für Infektionsmedizin e. V. (Berlin)
Analytik Jena AG (Jena) APOSAN Dr. Künzer GmbH (Cologne) Astellas Pharma GmbH (Munich) BD Biosciences (Heidelberg) BD Diagnostics (Heidelberg) BioLegend GmbH (Fell) BioMérieux Deutschland GmbH (Nürtingen)
Bristol‐Myers Squibb GmbH & Co. KGaA (Munich)
CeMeT GmbH (Tübingen) Cepheid GmbH (Frankfurt a. M.) Curetis AG (Holzgerlingen) Deutsche Gesellschaft für Immunologie e. V. (Marburg/Lahn)
Deutsche Gesellschaft für Infektiologie (Berlin)
Deutsches Zentrum für Infektionsforschung e. V. (Braunschweig)
Gilead Sciences GmbH (Martinsried) Infectopharm Arzneimittel (Heppenheim)
Janssen‐Cilag GmbH (Neuss) Leibniz‐Institut DSMZ (Braunschweig) Lophius Biosciences GmbH (Regensburg)
MSD Sharp & Dohme GmbH (Haar) New England BioLabs GmbH (Frankfurt a. M.)
OLS OMNI Life Science GmbH & Co. KG (Bremen)
PROMEGA GmbH (Mannheim) RIEMSER Pharma GmbH (Greifswald) TecoMedical GmbH (Bünde) ViiV Healthcare GmbH (Munich) XIV Booth number
7
4
6
19
21
17
16
26
11
10
9
25
13
12
4
4
23
18
2
1
14
8
20
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5
Tough
Hohe genetische Resistenzbarriere und
überzeugende virologische Wirksamkeit 1, 2
Forgiving
Gute Ansprechraten, selbst bei
suboptimal adhärenten Patienten *, 3
Reliable
Über 8 Jahre Praxiserfahrung – für eine
bewährte und verlässliche Therapie**
Bei der abgebildeten Person handelt es sich um ein Modell.
* ≤95% Adhärenz **PREZISTA® wurde im Februar 2007 von der EMEA zugelassen. 1 Lathouwers E, et al. Week 192 resistance analysis of HIV-1-infected, treatment-naive patients with virological failure in ARTEMIS, poster presented 9th European Workshop
on HIV & Hepatitis Treatment Strategies & Antiviral Drug Resistance, Paphos, Cyprus, March 23–25, 2011. Abstract O–07. 2 Orkin C, et al. Final 192-week efficacy and safety of once-daily darunavir/ritonavir compared with lopinavir/ritonavir in HIV-1-infected
treatment-naive patients in the ARTEMIS trial. HIV Med. 2013 Jan;14(1):49–59. 3 Nelson M et al. Suboptimal adherence to darunavir/ritonavir has minimal effect on efficacy compared with lopinavir/ritonavir in treatment-naive, HIV-infected patients: 96 week
ARTEMIS data J Antimicrob Chemother 2010;65(7):1505–09.
PREZISTA® 75 mg/- 150 mg/- 400 mg/- 600 mg/- 800 mg Filmtabletten/- 100 mg/ml Suspension zum
Einnehmen. Wirkstoff: Darunavir. Zusammensetz.: Filmtbl.: 1 Filmtabl. enth. 75 mg, 150 mg, 400 mg, 600 mg bzw.
800 mg Darunavir (als Ethanolat). Sonst. Bestandt.: Jede Tabl. enth. 0,834 mg (400 mg Tabl.) bzw. 2,750 mg (600 mg
Tabl.) Gelborange S (E110), mikrokristall. Cellulose, hochdisperses Siliciumdioxid, Crospovidon, Magnesiumstearat,
Hypromellose (800 mg Tabl.), Polyvinylalkohol – teilhydrolysiert, Macrogol 3350, Titandioxid (E171), Talkum. Suspension:
Jeder ml d. Susp. enth. 100 mg Darunavir (als Ethanolat). Sonst. Bestandt.: Hyprolose, mikrokrist. Cellulose, CarmelloseNatrium, Citronensäure-Monohydrat, Sucralose, Erdbeer-Sahne-Aroma, maskier. Aroma, Natrium-Methyl 4-hydroxybenzoat
(E219) 3,43 mg/ml, Salzsäure (zur pH Wert-Einstellung), ger. Wasser. Anw.geb.: Zusammen m. niedrig dosiertem Ritonavir
(rtv) in Kombination m. and. antiretrovir. Arzneim. zur Therapie v. Pat. m. Infekt. m. HIV-1. B. antiretroviral nicht vorbeh. Pat.
(400 mg, 800 mg, Susp.).B. antiretrov. vorbeh. Erw., einschl. derer, d. mehrf. vorbeh. wurden (75 mg, 150 mg, 400 mg, 600
mg, 800 mg, Susp.). B. pädiatr. Pat. ab 3 J. u. mind. 15 kg KG (75 mg, 150 mg, 600 mg, Susp.) bzw. ab 12 J. u. mind. 40
kg KG (400 mg, 800 mg). Zus. m. Cobicistat in Kombination m. and. antiretrovir. Arzneim. zur Therapie v. erwachs. Pat. m.
Infekt. m. HIV-1 (400 mg, 800 mg, Susp.). Dos.empf. s. jew. Fachinfo. Gegenanz.: Überempfindl. gg. Darunavir od. ein.
sonst. Bestandt.; schw. Leberfunkt.störg. (Child-Pugh-Klasse C). Zutreff. f. Darunavir i. Kombination m. Ritonavir od. Cobicistat:
gleichzeitige Anw. v. Rifampicin, Quetiapin, dem Kombinat.präp. Lopinavir/Ritonavir, Johanniskraut u. AM, deren Clearance in
hohem Maße v. CYP3A abhängig ist u. bei denen erhöh. Plasmakonz. m. schwerwieg. u./od. lebensbedrohl. Ereign.
einhergehen. Zutreff. f. Darunavir + Cobicistat: Anw. m. starken CYP3A4 Induktoren, wie z. B. Carbamazepin, Phenibarbital u.
Phenytoin, da diese d. Exposit gg. Darunavir u. Cobicistat reduz. könnten. Wg. Unsicherheiten bzgl. d. Entwicklungsgr. d.
Blut-Hirn-Schranke u. d. Leberenzyme b. Menschen ist PREZISTA® m. niedrig dos. Ritonavir nicht b. pädiatr. Pat. unter 3 J.
od. weniger als 15 kg KG anzuw.. Stillz.. Bes. Warnhinw. u. Vorsichtsmaßn.: Regelm. Überprüfg. d. virol. Ansprechens
empf., b. Fehlen od. Verlust Resistenztest durchführen. B. ART-vorbeh. Pat. m. einer od. mehr. DRV-RAMs od. ≥ 100.000
HIV-1-RNA Kopien/ml im Plasma od. einer CD4+-Zellzahl v. < 100 x 10 6 Zellen/l sollte PREZISTA® in Kombination m.
Cobicistat od. niedrig dosiertem Ritonavir nicht angew. werden. Bhdlgs.abbruch b. schweren Hautreakt.; Hautausschlag b.
ART-vorbeh. Pat. häufiger b. Komb.therapie m. Raltegravir. Vor u. währ. d. Bhdlg. Laborunters. d. Leberfunkt., insbes. b. Pat.
m. chron. Hepatitis, Leberzirrh. od. b. Pat. m. initialer Transaminasenerhöhung. B. neu auftr. Leberfunkt.störg. od. Verschlecht.
Unterbrech. od. Abbruch d. Bhdlg. erwägen. Vorsicht b.: leichter od. mäßiger Leberfunkts.störg. (Child-Pugh-Klasse A u. B),
chron. Hep. B u. C, Alter über 65 J.; Sulfonamidallerg.; Hämophilie, Diab. mellitus/Hyperglyk.; Schwangersch. nur bei
ausdrückl. Verordng.. Möglichk. e. Immunrekonstitutionssndr.. Über lebensbedrohl. u. tödl. Interakt. wurde b. Pat. berichtet,
die m. Colchicin u. starken Inhibit. v. CYP3A4 u. P Glykoprotein bhdlt. wurden. Efavirenz in Komb. m. PREZISTA®/Ritonavir
800/100 mg 1x tgl. kann zu suboptimalen Darunavir Cmin führen, daher b. Komb. m. Efavirenz Dosierung v. PREZISTA®/rtv
600/100 mg 2x tgl.. B. Wechsel d. pharmakokin. Verstärkers v. Ritonavir zu Cobicistat ist währ. d. ersten zwei Wo. d. Bhdlg.
m. Darunavir/Cobicistat Vors. geboten, besond. wenn währ. d. Anw. v. Ritonavir d. Dosier. v. gleichz. angew. Arzneim. titriert
od. eingestellt wurden. In diesen Fällen kann eine Dosisred. d. gleichz. angew. Arzneim. notw. sein. B. dialysepflicht. Pat.
wurde Cobicistat nicht untersucht. Cobicistat senkt d. geschätzte Creatinin-Clearance durch Hemmung d. tubul. Sekretion.
Nebenwirk.: Erwachs. Pat.: Darunavir/Ritonavir: Sehr häufig: Diarrhö. Häufig: Kopfschmerz, Erbrechen, Übelkeit,
Bauchschm., Alaninaminotransferase erhöht, erhöhte Amylase i. Blut, Hautausschlag (inkl. makulärer, makulopapul., papul.,
erythemat. u. juckender Ausschlag), Pruritus, Hypertriglycerid., Hypercholesterin., Hyperlipid., Diab. mell., periph. Neuropathie,
Schwindel, aufgeblähter Bauch, Dyspepsie, Flatulenz, Lipohyperthroph., Lipodystroph., Lipoatroph., Asthenie, Ermüdung
(Fatigue), Schlaflosigkeit. Gelegentlich: Myokardinfarkt, Angina pect., im EKG verläng. QT-Intervall, Tachykardie,
1502050_AZ_Hero_Berlin_A4.indd 1
Thrombozytopenie, Neutropenie, Leukopenie, Anämie, Lethargie, Parästhesie, Hypästhesie, Schläfrigk., konjunkt. Hyperämie,
trockenes Auge, Drehschwindel, Dyspnoe, Husten, Epistaxis, Reizungen i. Rachen, Pankreatitis, Gastritis, gastroösophag.
Refluxkrankheit, aphtöse Stomatitis, Würgereiz, Mundtrockenh., Aufstoßen, Empfindungsstörung im Mund, abdominelle
Beschwerden, Obstipat., erhöhte Lipase, (akutes) Nierenvers., Nephrolithiasis, erhöhtes Kreatinin i. Blut, Proteinurie,
Bilirubinurie, Dysurie, Nykturie, Pollakisurie, Angioödem, generalis. Hautausschlag, allerg. Dermatitis, Ekzem, Erythem, Akne,
trockene Haut, Nagelpigmentierung, Urtikaria, Hyperhidrose, Nachtschweiß, Alopezie, Myalgie, Osteonekrose, Muskelspasmen,
Muskelschwäche, Arthralgie, Extremitätenschmerz., Osteoporose, erhöhte Kreatinin-Phosphokinase i. Blut, Insulinresistenz,
Polydipsie, Gicht, Anorexie, Gewichtsabnahme, Gewichtszunahme, Hyperglykämie, Hypertonie, Pyrexie, Thoraxschmerz,
periph. Ödem, Hitzegefühl, Reizbark., Schmerz, allg. Unwohlsein, Immunrekonstitutionssyndr., (Arzneimittel-)Überempf.keit,
Hepatitis, zytolyt. Hepatitis, Steatosis hepatis, Transaminasen erhöht, Hepatomegalie, Bilirubin im Blut erhöht, alk. Phosph. im
Blut erhöht, Gammaglutamyltransferase erhöht, Aspartataminotransferase erhöht, erekt. Dysfunkt., Gynäkomastie, Depression,
Desorientierth., Angstzust., Schlafstörg., anomale Träume, Hypothyreose, TSH-Blutspiegel erhöht, vermind. Appetit,
vermehrter Appetit, vermind. HDL, Lactatdehydrogenase im Blut erhöht, Alpträume, vermind. Libido, Herpes simplex,
Dysgeusie, Aufmerksamkeitsstörg., Einschränkg. d. Gedächtnisleistung, Erröten. Selten: Eosinophilie, muskuloskelettale
Steifheit, Arthritis, Gelenksteifigkeit, Erythema multiforme, DRESS, Stevens-Johnson-Syndrom, Dermatitis, seborrh.
Dermatitis, Hautläsionen, Xerodermie, Verwirrth.zust., Stimmungsveränd., Unruhe, Synkope, Krampfanfall, Ageusie, Storg.
d. Schlafrhythm., Sehstörg., akuter Myokardinfarkt, Sinusbradykardie, Palpitationen, Rhinorrhö, Stomatitis, Hämatemesis,
Cheilitis, trock. Lippen, belegte Zunge, vermid. renale Kreatinin-Clearance, Schüttelfrost, anomales Gefühl, Xerosis. Nicht
bekannt: Toxisch Epidermale Nekrolyse, generalis. exanthemat. Pustulose. Erwachs. Pat.: Darunavir/Cobicistat:
Sehr häufig: Kopfschm., Diarrhö, Übelk., Hautausschlag (inkl. makul., makulopapulär. papul., erythem., juckend.,
general. Ausschlag u. allerg. Dermat.). Häufig: Überemp., Lipiddistroph. (einschl. Lipohypertroph., Lipiddistroph.,
Lipoatroph.)*, Anorexie, Diabetes mell., Hypercholesterin., Hypertriglycerid., Hyperlipid., anomale Träume, Erbr.,
Bauchschm., aufgebläh. Bauch, Dyspepsie, Flatulenz, Pankreasenzyme erhöht, Leberenzym. erhöht Angioödem, Pruritus,
Urtikaria, Myalgie, Osteonekrose*, Ermüdung, Serumkreatinin erhöht. Gelegentlich: Immunrekonstitutionssyndr., akute
Pankreatitis, Hepatitis*, zytolyt. Hepatitis*, Gynäkomastie*, Asthenie. Selten: DRESS*, Steven-Johnson-Syndr.*. Nicht
bekannt: Tox. epiderm. Nekrolyse*, akute general. exanthemat. Pustulose*. *: D. Nebenwirk. wurden nicht b. klin. Stud. m.
Darunavir/Cobicistat berichtet, aber bei d. Bhdlg. mit Darunavir/Ritonavir beob., so dass sie auch m. Darunavir/Cobicistat
erwartet werd. können. Zusätzl. b. antiretrov. Komb.therapie: Stoffwechsel-störg. (insbes. m. NRTIs): Myositis,
Myalgie, CPK-Wert-Erhöhung, selten Rhabdomyolyse.
Berichte v. Spontanblutg. b. Hämophilie-Pat.. Pädiatr.
Pat.: Sicherheitsdaten v. Phase-II-Studien zeigten b.
pädiatr. Pat. ein vergleichb. Sicherheitsprofil m. dem d.
Erwachs.population. Filmtbl.: Enth. Gelborange S (E110)
(nur 400 mg, 600 mg), das allerg. Reakt. hervorr.
kann. Suspension: Enth. Natrium-Methyl 4 hydroxybenzoat, was allerg. Reakt. auslösen kann (manchm. verzögert). Stand d. Inform.: 10/14. Verschreibungspflichtig.
Janssen-Cilag International NV, Beerse, Belgien.
JANSSEN-CILAG GmbH, 41457 Neuss.
07.10.15 17:27
Symposium I – Emerging Infections and Pneumonia SPEED TALKS ST 1 Surveillance and Outbreak Response Management and Analysis (SORMAS) to support the control of the Ebola Virus Disease (EVD) Outbreak C. Ameh1, S. Mall1, O. Adeoye1, J. Brauer1, S. Beermann1, J. Benzler1, H. Claus1, K. Denecke1, E. Enders1, E. Ilori1 M. Janke1, G. Kirchner1, T. Laedtke1, M. Lamshöft1, D. Moyer1, C. Nelson1, O. Ojo1, K. Pavle1, C. Perscheid1 M. Schapranow1, N. Schwarz1, D. Tom‐Aba1, S. Yennan1, G. Poggensee1, G. Krause1,2 1
2
Helmholtzzentrum für Infektionsforschung, Epidemiologie, Braunschweig, Germany Medizinische Hochschule Hannover, Hannover, Germany Objectives During the Ebola Virus Disease (EVD) outbreak in West Africa, contact tracing, information transfer and task management posed serious challenges. We developed the Surveillance and Outbreak Response Management and Analysis System (SORMAS) for real‐time surveillance, verification of cases and contact tracing. Methodology: We derived requirements for a holistic system by analyzing all procedures that were implemented by the Nigerian Ebola Emergency Operations Centre (EOC) during the EVD outbreak in Nigeria in August 2014 and the the Integrated Disease Surveillance and Response (IDSR) using the Design Thinking (DT) methodology. We developed a process model with seven personas representing the different tasks and responsibilities of EVD outbreak control. SORMAS interconnects mobile phones and tablet computers in the field with a cloud based in‐memory database system enabling real‐time situation assessment and bi‐directional information exchange. After a table top prototype test in February 2015 we conducted a four week pretest and five week pilot test in July and August involving 32 private and 32 public hospitals in 16 randomly selected local government administrations in Oyo and in Kano state in Nigeria. During the pilot we ran an agent‐based computer model simulating an EVD outbreak and generating realistic event injects for all SORMAS users. Additionally all real cholera, measles and avian influenza cases were processed using SORMAS. Results We identified the following 7 personas for which individual interphases and processes were implemented in SORMAS: Contact officers, surveillance officers and hospital informants worked smart phone based mobile interphases, while case officers, rumor managers, surveillance supervisors and contact supervisors used desk top based interphases. Quantitative analysis is still under way. Qualitative evaluation of the field pilot showed that interactive multi‐directional information exchange within the SORMAS outbreak management process works under field conditions. The user interfaces worked particularly well for the contact officers, whilst the interfaces for rumor officers and surveillance officers need improvements. The acceptability among field officers and state epidemiologists was very high. Mobile phone connectivity required the use of several mobile telecommunication providers in parallel. Conclusion The conceptual approach and the technical implementation of SORMAS proved to be functional under field conditions in Nigeria for routine surveillance as well as for complex management procedures of a simulated EVD outbreak. Results of quantitative analyses of the pilot will be fed into an improved design of SORMAS and development of additional functionalities, diseases and country settings. 1 Symposium I – Emerging Infections and Pneumonia ST 2 Safety and reactogenicity of rVSV Vaccine Expressing Zaire Ebola virus Glycoprotein: Phase I Trial in Germany C. Dahlke1,2, M. Zinser1, R. Kasonta1,2, V. Krähling3,4, A. Nolting1,2, H. C. Stubbe1,2, N. Biedenkopf3,4 M. Eickmann3,4, S. K. Fehling3,4, T. Strecker3,4, S. Borregard5, A. Jambrecina5, F. R. Stahl6, S. Becker3,4 A. W. Lohse1, S. Schmiedel1, M. M. Addo1,2 1
Universitätsklinikum Hamburg Eppendorf, Zentrum für Innere Medizin I, Hamburg, Germany German Center for Infection Research, partner site Hamburg‐Lübeck‐Borstel, Germany 3
Philipps University Marburg, Insitute for Virology, Marburg, Germany 4
German Center for Infection Research, partner site Giessen‐Marburg‐Langen, Germany 5
Clinical Trial Center North, Hamburg, Germany 6
Universitätsklinikum Hamburg Eppendorf, Institut für Klinische Chemie und Laboratoriumsmedizin, Hamburg, Germany 2
The magnitude of the ongoing Ebola epidemic as well as the high number of survivors with post‐Ebola syndrom demonstrate the urgent need for a vaccine against Ebola virus disease. The recombinant, attenuated vaccine rVSVΔG‐ZEBOV‐GP represents one promising candidate, which encodes for the immunogenic Zaire Ebola virus glycoprotein (ZEBOV‐GP). Several studies revealed both long‐term protection in non‐human primates and the potential for post‐exposure prophylaxis after a single injection. As part of the WHO led VSV Ebola Consortium (VEBCON), we performed an open‐label, dose escalation phase I trial to assess safety, tolerability and immunogenicity of rVSVΔG‐ZEBOV‐GP (NCT02283099) at the University Medical Center Hamburg‐Eppendorf. Thirty healthy adults were vaccinated with 3x105, 3x106 or 2x107 plaque‐
forming units (pfu) (10 subjects per group). The vaccine was well tolerated without major safety concerns. The reported adverse events were mostly mild. Vaccinees developed changes in blood counts, like asymptomatic lymphopenia and monocytosis. Low‐level viremia was dose‐dependent and identified within three days in all participants immunized with ≥ 3x106 pfu; there was no detectable shedding into urine or saliva. Antigen‐specific T cell responses were overall found to be of low magnitude. All analysed participants developed ZEBOV‐GP specific antibody responses, including neutralizing activity in most ‐ providing evidence of good immunogenicity. These data provide safety, reactogenicity and immunogenicity signals for rVSVΔG‐ZEBOV‐GP in humans1. The results outlined here guided the dose selection for the phase II/III efficacy trials in Africa, which demonstrate highly promising results in the Ebola ça Suffit trial in Guinea2. Furthermore, our results provide important safety data for the rVSV platform, which may represent a powerful tool for novel vaccine strategies against emerging viruses. 1. Agnandji ST et al., Phase 1 Trials of rVSV Ebola Vaccine in Africa and Europe — Preliminary Report, 2014, NEJM. 2. Henao‐Restrepo et al., Efficacy and effectiveness of an rVSV‐vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster‐randomised trial, 2015, The Lancet. 2 Symposium I – Emerging Infections and Pneumonia ST 3 Building an “Ebola aid‐workers´ School” in one week ‐ Effective and rapid set‐up of the pre‐deployment training program for >200 staff for the Ebola Intervention in West Africa, 2014. Wuerzburg, Germany, 2014. M. Gertler1,2, A. Matuschek1, M. Di Gennaro3, S. Loik4, C. Kleine5, J. Paul1, M. Roth1, N. Gresser1, A. Fabricius3 J. Butenop1, A. Stich1 1
Medical Mission Institute, Wuerzburg, Germany Médecins Sans Frontières, Berlin, Germany 3
German Red Cross, Berlin, Germany 4
German Armed Forces, Joint Support Services, Bonn, Germany 5
Department of Infectious Diseases & Tropical Medicine, Center for Internal Medicine II, University Hospital Frankfurt, Frankfurt a. M., Germany 2
Introduction By end September 2014, the German Red Cross (GRC) was mandated by the German Government to support the international Ebola intervention in West‐Africa. GRC requested specific training of the Medical Mission Institute Wuerzburg (MI) as a mandatory prerequesite for delegates being deployed to Liberia and Sierra Leone. MI ‐ supported by Médecins Sans Frontières (MSF) ‐ initiated an Ebola predeployment training. Here, we describe initiation, progress and challenges of the program, the participants and their course evaluation. Methods MI contributed the complete training set‐up and ‐ together with MSF ‐ expertise in clinical care and epidemic response regarding Ebola. MSF contributed a course concept and supported recruitement of trainers. The training curriculum included lectures and practicing of logistic and clinical procedures as applied in MSF Ebola projects guided by field‐experienced trainers in a simulated Ebola Treatment Unit (sETU). Course evaluation was initially based on protocolled and regular but not systematic feed‐back discussions with participants and later (5 last sessions) on a standardised questionnaire with 12 questions regarding lectures and practical exercises (rating range: 1 „excellent“ to 5 „poor“). Results Training preparation started 26 September 2014. From 6 October until 24 February 2015, 214 trainees participated in 14 two‐day courses in a newly‐constructed sETU in Wuerzburg. With an average trainer‐trainee ratio of 1:3 (range 1:2.3 to 1:4.6) participants trained standardised procedures e.g. (un‐)dressing of personal protective equipment, taking blood samples, handling corpses and waste. GRC sent 166 (78%), German Armed Forces (GAF) 12 (6%) and others 36 (16%) participants. Of 96 GRC staff deployed to West Africa, 90 (94%) participated in the training. The first trainees left to the field on 8 October. In returned evaluation sheets (68/86; 79%), 706/757 (93%) ratings were either „excellent“ or „good“. A demand for more practical exercises and an appreciation of learning directly from field‐experienced Ebola aid workers was most frequently stated. Discussion The pre‐existing training capacity for highly contagious diseases in Wuerzburg, MSF´s Ebola‐guidelines and field‐experienced trainers plus pragmatic cooperation were key to rapid and effective implementation and positive evaluation of the training and thus the joint GRC/GAF intervention. Formal evaluation of only 36% of trainings limits the success assessment. However, we recommend for similar training needs: pragmatic inter‐
organisation cooperation, use of approved resources, guidelines and hands‐on experience of trainers for small‐
group practicals. Proportion of lecture time might be reduced through obligatory preceding literature study or e‐learning tools. The achieved training capacities should be conserved for future emergencies. Systematic training evaluation must be available from the beginning. 3 Symposium I – Emerging Infections and Pneumonia ST 4 Randomized phase I/IIa clinical trial demonstrates safety and immunogenicity of an MVA‐
based influenza A/H5N1 vaccine in healthy adults G. Sutter1, J. Kreijtz2, M. Goeijenbier2, F. M. Moesker2, L. van den Dries2, A. Volz1, M. Lehmann1 S. Goeijenbier2, H. L. De Gruyter2, G. de Mutsert2, D. A. van de Vijver2, R. F. Fouchier2, E. C. V. van Gorp2 G. F. Rimmelzwaan2, A. D. Osterhaus2 1
2
LMU Munich, Institut für Infektionsmedizin und Zoonosen, Munich, Germany Erasmus MC, Rotterdam, The Netherlands Background Modified Vaccinia virus Ankara is a potent viral vector platform for H5N1 influenza vaccine development. Preclinical evaluation of MVA‐based H5N1 vaccines showed their immunogenicity and safety in various animal models, warranting clinical evaluation. Methods In this randomized double‐blind phase I/IIa study young healthy volunteers were immunized once or twice with a normal dose or a tenfold lower dose of either the MVA‐H5‐sfMR or MVA‐F6‐sfMR vaccine. Subjects that received the MVA‐H5‐sfMR vaccine were eligible for a boost immunization one year after the first immunization. Primary safety endpoints were local and/or systemic reactions. Secondary outcomes were hemagglutination inhibition (HI) and virus‐neutralization (VN) antibody titers in the sera from the subjects. The trial is registered at the Dutch Trial Register (www.trialregister.nl) (NTR registration number: NTR3401). Findings 79 of the 80 subjects that were enrolled completed the study. No serious adverse events occurred. The majority of the subjects experienced one or more local and systemic reactions. Subjects that received the tenfold lower dose were prone to develop less systemic reactions. The MVA‐H5‐sfMR 108 pfu vaccine induced significantly higher antibody responses. 27 of the 39 eligible subjects were enrolled in the boost immunization study. The results indicated that a single shot MVA‐H5‐sfMR prime immunizations resulted in higher antibody responses upon boost immunization than two shots with MVA‐H5‐sfMR given in a conventional four‐week interval. Interpretation The data generated by our study strongly suggest the potency of the MVA‐based pandemic H5N1 vaccine and underline that vaccine candidates arising from the MVA platform hold great promise for the future. 4 Symposium I – Emerging Infections and Pneumonia ST 5 Murine bone marrow‐derived mesenchymal stem cells exert anti‐viral and barrier‐
protective properties in influenza virus infection L. Jankauskaite1, C. Schmoldt1, J. Lohmeyer1, S. Herold1 1
UGMLC, Giessen, Germany Introduction Influenza virus (IV) infects the upper respiratory tract and occasionally spreads to the alveolar compartment causing primary IV pneumonia. This frequently progresses to acute respiratory distress syndrome (ARDS). Antiviral therapies are only effective in the very beginning of infection and specific treatment strategies for IV‐
induced ARDS are lacking. Recently, mesenchymal stem cells (MSC) were attributed a beneficial role in acute lung injury induced by bacterial infections. Objectives To identify, isolate, characterize and amplify primary murine bone marrow MSC (mBM‐MSC). To characterize MSC therapeutic potential in IV‐induced lung injury. Materials and Methods: MSC were isolated by flow‐cytometry (FACS) from mice bone marrow and cultivated in vitro. mBM‐MSC expression markers and stem cell properties were characterized by FACS and microscopy. mBM‐MSC were co‐
cultured with IV‐infected primary murine alveolar epithelia cells (AECs) which were subsequently submitted to genome array and FACS analysis. mBM‐MSC were injected intratracheally to IV‐infected C57Bl6 mice and their impact on alveolar damage was quantified by microscopy and FACS measurements. Results Generated mBM‐MSC expressed stem cell‐specific markers and demonstrated differentiation potential. Genome array analysis revealed a strong up‐regulation of genes involved in proliferation (cell division, cyclins), in interferon signalling (ISGs) and in virus resistance (Mx2, Bst2/Tetherin) in infected AECs cultured with mBM‐
MSC compared to infected AEC in monoculture. In ex vivo experiments mBM‐MSC as well as their conditioned media strongly diminished IV replication, increased regeneration and consequently decreased IV‐induced apoptosis in AEC. This paracrine action of the mBM‐MSC was associated with up‐regulation of type I interferons of the anti‐apoptotic factor stanniocalcin‐1 and of growth factors FGF10 (fibroblast growth factor 10) and VEGF (vascular endothelial growth factor). In vivo, intratracheal instillation of mBM‐MSC after IV challenge strongly increased virus clearance, decreased alveolar injury and was associated with better outcome. Of note, mBM‐
MSCs also increased the regenerative response of the epithelial stem/progenitor cell pool of the distal lung in vivo. Conclusions Our ex vivo as well as our in vivo experiments show a beneficial role of mBM‐MSCs in IV pneumonia confirming their therapeutic potential in IV‐induced lung injury. 5 Symposium II – Infections of the immunocompromised Host: Nosocomial and Gastrointestinal Infections ST 6 Chlorhexidine containing iv‐catheter securement dressings for the prevention of central venous catheter‐related bloodstream infections in neutropenic patients: a randomized trial (COAT study) L. M. Biehl1,2, A. Huth1, J. Panse3, M. Hentrich4, M. Engelhardt5, K. Schäfer‐Eckart6, G. Kofla7, M. Kiehl8 C. Wendtner9, M. Karthaus10, M. Hellmich11, H. Christ11, O. A. Cornely1,12,13,14, M. J. G. Vehreschild1,2 1
University Hospital of Cologne, Department I of Internal Medicine, Cologne, Germany partner site Bonn‐Cologne, German Centre for Infection Research (DZIF), Cologne, Germany 3
RWTH Aachen University Hospital, Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Aachen, Germany 4
Red Cross Hospital Munich, Department of Medicine III, Munich, Germany 5
University of Freiburg, Department of Medicine I, Hematology, Oncology and Stem Cell Transplantation, Medical Center, Freiburg im Breisgau, Germany 6
Klinikum Nürnberg, Medical Clinic 5, Hematology and Oncology, Nürnberg, Germany 7
Charitè Campus Mitte, Charité University Medicine, Department of Medicine, Division of Oncology/ Hematology, Berlin, Germany 8
Clinical Center Frankfurt/Oder, Medical Clinic I, Hematology and medical Oncology, Hemostaseology, Frankfurt/Oder, Germany 9
Klinikum Schwabing, Department of Haematology, Oncology, Immunology, Palliative Care, Infectious Diseases and Tropical Medicine, Munich, Germany 10
Klinikum Neuperlach, Department of Hematology and Oncology, Munich, Germany 11
University of Cologne, Institute of Medical Statistics, Informatics and Epidemiology, Cologne, Germany 12
University of Cologne, Clinical Trials Centre Cologne, ZKS Cologne, BMBF 01KN1106, Cologne, Germany 13
University of Cologne, Center for Integrated Oncology CIO Cologne/Bonn, Cologne, Germany 14
University of Cologne, Cologne Excellence Cluster on Cellular Stress Responses in Aging‐Associated Diseases (CECAD), Cologne, Germany 2
Introduction Central venous catheter‐related bloodstream infections (CRBSI) are a frequent cause of morbidity and mortality in patients with chemotherapy‐induced neutropenia. Chlorhexidine containing iv‐catheter securement dressings may prevent CRBSI. Methods A multicenter randomized controlled trial was conducted at 10 German hematology departments. We compared chlorhexidine containing highly adhesive dressings with non‐chlorhexidine highly adhesive dressings (control) in high‐risk neutropenic patients. The primary endpoint was the incidence of definite CRBSI within the first 14 days (dCRBSI14) of central venous catheter (CVC) placement. Secondary endpoints included overall incidence of definite CRBSI (dCRBSI), 14 days and overall incidence of definite or probable CRBSI (dpCRBSI14 and dpCRBSI) and incidence of unscheduled dressing changes and adverse events. CRBSI were defined according to the Guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Oncology (DGHO) from 2008. Results From Feb 2012 to Sept 2014, 630 patients were included in the study. 17 patients were excluded from evaluation. Incidence of dCRBSI14 was 2.6% (8/307) in the chlorhexidine and 3.9% (12/306) in the control group (p=0.375), and for dCRBSI 4.2% (13/307) vs. 7.9% (24/306), respectively (p=0.064). Both dpCBRSI14 and dpCRBSI were significantly less frequent in the study group with dpCRBSI14 in 6.5% (20/307) of the chlorhexidine group as compared to 11% (34/306) in the control group (p=0.047), and dpCRBSI in 10.4% (32/307) vs. 17% (52/306), respectively (p=0.019). Dressing intolerance leading to discontinuation was similar between both groups (7.2% and 7.8%). Conclusion The application of chlorhexidine containing iv‐catheter securement dressings reduces the incidence of definite or probable CRBSI in neutropenic patients. Clinical trial registered with Clinicaltrials.gov (NCT01544686). 6 Symposium II – Infections of the immunocompromised Host: Nosocomial and Gastrointestinal Infections ST 7 Intestinal sugars modulate microbiota composition and the susceptibility to enteric pathogens A. Suwandi1, P. Rausch2,3, N. Steck3, J. Seidel3, M. Basic4, A. Bleich4, J. Johnsen5, J. Baines2,3, G. Grassl1 1
Medizinische Hochschule Hannover, Institut für Medizinische Mikrobiologie, Hannover, Germany Max Planck Institute for Evolutionary Biology, Plön, Germany 3
University of Kiel, Kiel, Germany 4
Medizinische Hochschule Hannover, Hannover, Germany 5
Puget Sound Blood Center, Seattle, USA 2
Glycans on mucosal surfaces play an important role in host‐microbe interactions. The B4galnt2 gene encodes a blood‐group‐related glycosyltransferase that is subject to strong selective forces in natural house mouse populations, whereby a common allelic variant exists that results in loss of B4galnt2 gene expression in the gastrointestinal tract. Loss of B4galnt2 expression is associated with altered intestinal microbiota. We hypothesized that altered glycan‐dependent intestinal host‐microbe interactions may underlie these signatures of selection. Here, we investigated whether B4galnt2 expression influences host susceptibility to the enteric pathogen Salmonella enterica serovar Typhimurium. Using in vivo and in vitro infections with S. Typhimurium we evaluated adhesion and invasion of the bacteria into epithelial cells. In addition, we analyzed histopathological changes and the activation of pro‐inflammatory host response. Microbiota composition was determined using 16S rRNA gene sequencing. Fecal transplantation experiments were employed to demonstrate the role of the genotype‐dependent microbiota. B4galnt2 intestinal expression was strongly associated with increased susceptibility to Salmonella Typhimurium as evidenced by increased intestinal pathology, elevated inflammatory cytokines and infiltrating immune cells. Fecal transfer experiments demonstrated a crucial role of the B4galnt2 dependent microbiota in conferring susceptibility to Salmonella infection. These data demonstrate a critical role for B4galnt2 in intestinal pathogen induced inflammation. We speculate that B4galnt2‐specific differences in host susceptibility to intestinal inflammation underlie the strong signatures of balancing selection observed at the B4galnt2 locus in wild mouse populations. 7 Symposium II – Infections of the immunocompromised Host: Nosocomial and Gastrointestinal Infections ST 8 Identification of a novel compound inhibiting the type III secretion system 1 of Salmonella Typhimurium S. Wagner1, I. Grin1, A. Poso2, M. Riess1 1
2
Universität Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, Tübingen, Germany Universität Tübingen, Internal Medicine I, Tübingen, Germany Resistance of bacteria is a serious emerging threat and hence the search for new antibiotics is of high priority. A novel class of antibiotics that block infection instead of killing bacteria is thought to exhibit a lower potential for the development of resistance because of a reduced selection pressure compared to conventional antibiotics. These antiinfectives target virulence mechanisms of pathogenic bacteria such as adhesion determinants or toxin delivery systems. Type III secretion systems of Gram‐negative bacteria, often enteropathogens, serve the injection of bacterial effector proteins into eukaryotic target cells to promote infection and colonization. Type III secretion systems are not only excellent targets for antiinfectives because the virulence of many pathogens depends on these systems but also because type III secretion systems are highly conserved and hence a single drug has the potential to act against a broad spectrum of enteropathogens. We identified drugable pockets in the cytoplasmic domain of the major export apparatus protein of the type III secretion system‐1 of Salmonella Typhimurium and ran an in silico screen with a commercially available compound library to identify binding compounds with a potential to inhibit type III secretion. Candidate compounds were tested by several in vivo assays for their effect on type III‐dependend secretion. One compound was identified that showed a strong inhibitory effect on type III secretion while leaving the viability of bacterial and eukaryotic cells unaffected. Besides exhibiting an instant inhibitory effect on type III secretion ‐ as one would expect for a compound acting on the major export apparatus protein ‐ the compound also strongly reduced the overall expression of pathogenicity island 1 encoding the type III secretion system under investigation. The chemical potential of the compound for the analysis of structure‐activity‐relationships as well as the observed dual effect on type III secretion make the identified compound a very promising candidate for hit to lead development and therefore it may once serve to prevent and cure gastrointestinal infections cause by enteropathogens. 8 S y m p o si u m
I I – nfections o f th e i m m u no c o m p ro m i sed H o st:
N o so c o m i al and astrointestinal I nf ec tions
S T 9
nduction o f p ro - and anti- in ammato ry h u m an T c el l f untio nalities b y di stinc t m i c ro b es
C . Zielinski1
1
Technische Universität München, Institut für Medizinische Mikrobiologie, I
unologie und Hygiene,
unich, Germany
Th17 cells have emerged as a new T helper cell lineage involved in the clearance of extracellular bacteria and
fungi and in autoi
unity.
We demonstrate that uman Th17 cells transiently produce the anti-infla
atory cytokine IL-10 upon
sti ulation. Interestingly, IL-10 expression was accompanied by reciprocal down-regulation of IL-17, leading to
a functional regulatory Th17 cell phenotype a er the peak of the effector response. Regulatory activities of
Th17 cells were demonstrated by their ability to suppress responder cell proliferation and by their ability to
downregulate pro-infla
atory cytokine production by innate cells. The ability of Th17 cells to express IL-10
was, however, restricted to certain antigen specificities. E x v iv o isolated C . alb ican s specific Th17 cells could not
produce IL-10 in comparison to S . aure us specific Th17 cells. This was due to differential priming re uirements
of these Th17 cell sub-populations. IL-1beta instructed naï ve T cells to develop into a pro-infla
atory nonIL10 expressing Th17 cell subset. Th17 cell priming with S . aure us , however, was not IL-1beta dependent,
leading instead to the generation of IL-10 producing Th17 cells with self-regulatory activities.
us, using a novel approach that combines the in v it ro priming of naive T cells by whole microbes with the e x
v iv o analysis of memory T cells, we were able to un ask the existence of two types of Th17 cells that differ in
priming re uirements, TC R repertoire and function.
Selektiv. Bakterizid. Anhaltend wirksam.*, 1, 2
Bei C. difficileInfektionen
DIFICLIR™
Unübertroffene Heilungsraten1-3
Reduktion des Rezidivrisikos um 46 %1-3
* im Vergleich zu Vancomycin signifikant höhere Raten anhaltender Heilung definiert als Abklingen der Diarrhö unter der Therapie
und kein Rezidiv innerhalb von 30 Tagen nach Beendigung der Therapie
1. Louie TJ et al. N Engl J Med 2011; 364(5): 422 – 431. 2. Cornely OA et al. Lancet Infect Dis 2012; 12: 281 – 289. 3. Crook DW et al. Clin Infect Dis 2012; 55(Suppl 2): 93 –103.
DIFICLIR® 200 mg Filmtabletten. Wirkstoff: Fidaxomicin. Zusammensetzung: Eine Filmtablette enthält Wirkstoff: 200 mg Fidaxomicin. Sonstige Bestandteile: Tablettenkern: Mikrokristalline Cellulose,
vorverkleisterte Stärke, Hydroxypropylcellulose, butyliertes Hydroxytoluol, Carboxymethylstärke-Natrium, Magnesiumstearat. Filmüberzug: Polyvinylalkohol, Titandioxid, Talkum, Macrogol, Lecithin (Soja).
Anwendungsgebiete: DIFICLIR® ist indiziert bei Erwachsenen zur Behandlung von Clostridium difficile-Infektionen (CDI), auch bekannt unter der Bezeichnung Clostridium difficile-assoziierte Diarrhö (CDAD).
Offizielle Leitlinien zum angemessenen Gebrauch von Antibiotika sollten berücksichtigt werden. Gegenanzeigen: Überempfindlichkeit gegen den Wirkstoff oder einen der sonstigen Bestandteile. Nebenwirkungen: Häufig (* 1/100, < 1/10): Erbrechen, Übelkeit, Obstipation. Gelegentlich (* 1/1.000, < 1/100): Hautausschlag, Juckreiz, Appetitabnahme, Schwindelgefühl, Kopfschmerz, Geschmacksstörung,
Völlegefühl, Flatulenz, Mundtrockenheit, Anstieg der Alaninaminotransferase. Häufigkeit auf Grundlage der verfügbaren Daten nicht abschätzbar: Überempfindlichkeitsreaktionen
(Angioödeme, Dyspnoe). Warnhinweise: Für Kinder unzugänglich aufbewahren. Verschreibungspflichtig. Weitere Einzelheiten enthalten die Fach- und Gebrauchsinformation.
Pharmazeutischer Unternehmer: Astellas Pharma Europe B.V., Sylviusweg 62, 2333 BE Leiden, Niederlande; Deutsche Vertretung des pharmazeutischen Unternehmers:
Astellas Pharma GmbH, Postfach 50 01 66, 80971 München. Stand: Juni 2014.
Symposium II – Infections of the immunocompromised Host: Nosocomial and Gastrointestinal Infections ST 10 Identification of T cell epitopes for a therapeutic HPV16 vaccine S. Hoppe1,2, J. P. Schessner2, L. Dressler2, J. Winter2, R. Blatnik1,2, A. Steinbach2, H. Khallouf2, M. Wühl2 A. Klevenz2, A. B. Riemer1,2 1
2
Deutsches Zentrum für Infektionsforschung (DZIF), Junior Group Molecular Vaccine Design, Heidelberg, Germany Deutsches Krebsforschungszentrum (DKFZ), Immuntherapie und ‐prävention, Heidelberg, Germany To rationally design therapeutic human papillomavirus (HPV) vaccines, it is important to determine which T cell epitopes are presented on HPV‐transformed cells. Due to viral immune evasion mechanism, not every epitope derived from viral proteins is presented by Human Leukocyte Antigen (HLA)‐molecules on the cancer cell surface. HPV16 has been identified as the causative agent in 50% of all cervical cancer cases and in approximately 95% of all extra‐cervical HPV‐induced tumors. The transforming potential of high‐risk HPVs is mediated by two consistently expressed viral oncoproteins, E6 and E7. As the induction and maintenance of the malignant phenotype depends on these two proteins, they represent ideal targets for cancer immunotherapy. A therapeutic vaccine that is applicable to everyone without prior HLA typing needs to contain epitopes for all HLA‐types. Definition of epitopes for the five major HLA supertypes allows >95% population coverage. Up to date, HPV T cell epitopes have mostly been determined for the most prevalent HLA type, HLA‐A2. We now aim to identify HPV16 E6 and E7 T cell epitopes for other common HLA supertypes, namely HLA‐A3, HLA‐
A11 and HLA‐A24. Various in silico algorithms were employed to predict prospective epitopes from the HPV16 E6 and E7 proteins for the mentioned HLA supertypes. Altogether, sixty‐four 8‐ to 11‐mer epitopes were predicted for HLA‐A3, ninety epitopes for HLA‐A11, and 115 epitopes for HLA‐A24. Fifteen previously known binding peptides were confirmed and ninety‐three novel binding peptides were identified in competition‐based binding assays. The presence of binding peptides on HPV16‐transformed cells is analyzed by mass spectrometry analysis of immunoprecipitated HLA‐peptide complexes. To determine immunogenicity of binding peptides, peptide‐
specific short term T cell lines were generated. To this end, peripheral blood mononuclear cells (PBMCs) from buffy coats with known HLA types were stimulated with previously identified HLA‐matching peptides and cultured for twelve days. Several peptides induced interferon gamma (IFNγ)‐responses. Additionally, peptide‐
specific long term T cell lines were generated for the four most promising candidate peptides from HLA‐A24 positive healthy donors. Functional assays, such as IFNγ‐ELISpot and cytotoxicity assays, determined the best vaccine candidates. In conclusion, we identified several new HPV16 E6 and E7 T cell epitopes. Verified epitopes are the basis of rational therapeutic vaccine design and also important for immunomonitoring purposes. 10 Symposium II – Infections of the immunocompromised Host: Nosocomial and Gastrointestinal Infections ST 11 Identification of novel mutations in Common Variable Immunodeficiency Syndrome (CVID) through targeted re‐sequencing of a CVID cohort F. Atschekzei1, C. Schröder1, T. Witte1, R. E. Schmidt1, B. Grimbacher2 1
2
MHH, Klinik für Immunologie und Rheumatologie, Hannover, Germany Centrum für Chronische Immundefizienz Universitätsklinikum, Freiburg, Germany Background CVID comprises a heterogeneous group of diseases characterized by a significant hypogammaglobulinemia of unknown cause, failure to produce specific antibodies after immunizations and susceptibility to bacterial infections, predominantly caused by encapsulated bacteria. Moreover CVID includes T‐lymphocyte abnormalities, which may partly explain the tendency to lymphoproliferative and autoimmune disorders seen in CVID patients. The majority of the genetic mechanisms leading to CVID are still unclear. Defects in the genes that encode for ICOS, TACI, CD19, BAFFR, CD81, CD20, and CD21 have been reported. The identification and analysis of these gene defects have led to novel hypothesis on the pathogenesis of CVID. However, gene defects have been identified in less than 10% of patients, and therefore they account only for a small part of the cases of CVID. Objective We sought to assess the genetic background of CVID‐ patients by targeted re‐sequencing of 45 CVID subjects. Material and methods genomic DNA was isolated from whole blood obtained 45 adult patients with CVID. To determine the nucleotide variations in 45 CVID subjects, we used a next‐generation sequencing panel for targeted re‐
sequencing, comprising 47 known and candidate genes associated with primary immune deficiencies. The detected mutations were validated by Sanger sequencing and interpreted on pathogenicity using bioinformatics tools (UniProt, UCSC genome browser, SIFT, PolyPhen, Gen Scan, etc.). Results In the present study we found novel mutations in LRBA‐ and RAG1 genes, among others. One CVID‐ patient, who was born to a consanguineous family, showed post‐mortem a homozygous mutation in LRBA gene, which altered the donor splice‐site (IVS6+5_8del.). This mutation may lead to frameshift or splice site disruption and could result in production of a nonfunctional protein. One patient was documented post‐mortem with a novel mutation (c.1430delC), which leads to a frameshift and a following premature stop signal 14 codons afterwards (p.F478Sfs*14), and two previously described mutations in RAG1 (p.H375D, p.R474C). The variant p.H375D occurred in cis with p.F478Sfs*14 and in trans with p.R474C. The frameshift p.F478Sfs*14 mutation represents a novel and not published mutation. The mRNA covers approximately half of the normal 1042 amino acids. Therefore we suggest that it may be prone to nonsense‐mediated decay. The variant p.H375D could lead to an altered binding of the zinc‐ion. Given the evidence that p.H375D destroys the zinc finger and c.1430delC causes truncation, these two may be more relevant for CVID than p.R474C. Conclusion Our study can be a further step to understand the genetic background of CVID patients and can be helpful to continuously refining diagnostic and therapeutic methods. Keywords LRBA (Lipopolysaccharide‐(LPS) responsive vesicle trafficking, beach and anchor containing), RAG‐1 (recombination‐activating gene) Supported by DZIF Project number: TTU 07.801, KFO 250 Project number: TPO3 11 Symposium III – Mycobacteria, Tuberculosis and Malaria ST 12 The role of filarial infections and filarial‐induced immunomodulation in the pathogenesis of epilepsy in sub‐Saharan Africa T. Wagner1,2,3, A. Hörauf1, K. Pfarr1, C. Prazeres da Costa2, A.‐ S. Winkler4 1
DZIF Bonn, Institute of Medical Microbiology, Immunology and Parasitology, University of Bonn, Bonn, Germany DZIF Munich, Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany 3
Centre for Pediatric and Adolescent Medicine, University Hospital Heidelberg, Heidelberg, Germany 4
Department of Neurology, Technische Universität München, Munich, Germany 2
Background Epilepsy seems more common in low income than in high income countries and may be associated with filarial infections such as Onchocerca volvulus, Mansonella streptocerca and Mansonella perstans. Some authors observed epilepsy to cluster in areas with fast flowing rivers where Simuliidae vectors breed and onchocerciasis is hyperendemic. Consistent therewith, skin snip microscopy, PCR and antibody tests confirmed a higher prevalence of filarial infections in people with epilepsy compared to health controls. Nevertheless it remains uncertain how this potential relationship is mediated, whether it is attributed to the parasite or the immune response and what impact recent onchocerciasis control programs might have. Study design and preliminary results We hypothesize that prevalence and quality of filarial infections as well as the host’s immune response may differ in people with epilepsy and healthy controls. With support of a DZIF clinical leave stipend we therefore developed a study to characterize filariae and their Wolbachia endosymbionts, investigate the activity of the filarial infection and Treg‐ driven immune response in Mahenge, Tanzania, an area with an extraordinary high epilepsy burden of up to 39 per 1000 inhabitants. Therefore we collected clinical data as well as blood, skin, urine and stool samples of 150 patients with different forms of epilepsy and in neurologically healthy endemic controls and are planning in depth laboratory work up at the DZIF sites Bonn and Munich. Preliminary skin snip data confirm that onchocerciasis is significantly more prevalent in people with epilepsy than in neurologically healthy controls (p<0.025). We will further characterize Onchocerca and Mansonella spp. strains found in patients with epilepsy and controls via PCR of whole blood samples and determine Wolbachia loads and its features. Furthermore, we intend to quantify the infection with O. volvulus from urine by measuring a novel unique biomarker, N‐acetyltyramine‐O,ß‐glucuronide (NATOG) and will correlate the findings with other markers that define the host’s immune response in PBMCs and serum phenotype (e.g. Th1/2/17 and Treg, pro‐ and anti‐inflammatory cytokines). Overall, the designated approach which relates different assays and clinical findings aims to elucidate quantity and quality of filarial infections and corresponding immune response in people with epilepsy and may provide explanations for the robust epidemiologic associations. 12 Symposium III – Mycobacteria, Tuberculosis and Malaria ST 13 Long‐term safety and efficacy results of the PanACEA MAMS‐TB randomised controlled trial N. Heinrich1,2,3, P. Phillips3,4, S. Rehal3,4, N. E. Ntinginya5,3, L. T. Minja3,6, K. Reither3,7, G. Kibiki3,8, I. Sanne3,9 K. Mellet3,9, A. Diacon3,9,10, R. Dawson3,11, G. Churchyard3,12, A. Nunn3,4, A. Colbers3,13, A.‐ M. Mekota1,2,3 R. Aarnoutse3,13, S. Gillespie3,14, M. Hoelscher1,2,3, M. Boeree3,13 1
Klinikum der Universität München, Abteilung für Infektionskrankheiten und Tropenmedizin, Munich, Germany Deutsches Zentrum für Infektionsforschung, Partner site Munich, Munich, Germany 3
on behalf of the PanACEA consortium, The Netherlands 4
University College of London, Medical Research Council ‐ Clinical Trials Unit, London, Great Britain 5
NIMR ‐ Mbeya Medical Research Centre, Mbeya, Tansania 6
Ifakara Health Institute, Bagamoyo Research and Training Center, Bagamoyo, Tanzania 7
Swiss Tropical and Public Health Institute, Basel, Switzerland 8
Kilimanjaro Clinical Research Institute, Moshi, Tanzania 9
University of the Witwatersrand, Clinical HIV Research Unit, Johannesburg, South Africa 10
University of Stellenbosch, MRC Centre for Molecular and Cellular Biology, Cape Town, South Africa 11
University of Cape Town, Lung Institute, Cape Town, South Africa 12
Aurum Institute for Health Research, Johannesburg, South Africa 13
Radboud University of Nijmegen Medical Center, Nijmegen, The Netherlands 14
University of St. Andrews, St. Andrews, Great Britain 2
Background The PanACEA MAMS‐TB trial was conducted in seven African sites to identify regimens that reduced time to culture conversion in liquid media. 12‐week results showed that 35mg/kg of rifampicin accelerated culture conversion. We now report on results to end of treatment and follow‐up. Methods Adult patients with smear‐positive TB were randomly allocated to 12 weeks of 1) 35mg/kg rifampicin (R), together with isoniazid (H), pyrazinamide (Z), and ethambutol (E), 2) SQ109 together with standard dose RZH 3) SQ109 and 20 mg/kg R with ZH, 4) moxifloxacin and 20mg/kg R with ZH, 5) a control arm of 8 weeks of standard dose HRZE. All patients then received standard RH to complete a total of 26 weeks of treatment. Post‐
treatment follow‐up was conducted at 3 months and 6 months by telephone with patients asked to attend the clinic for assessment if feeling unwell. Results Results of culture conversion to 26 weeks were consistent with 12‐week results. 35mg/kg of R shortened culture conversion from a median of 62 days (control) to 48 days. 308(85%)/363 patients completed post‐
treatment follow‐up to 6 months. 34(9%) patients were found to be unwell at 3 or 6 months, of whom 33 attended a clinic with only 7(2%) confirmed relapses. One patient acquired MDR‐TB, and one patient died shortly before completing continuation treatment. Hepatic adverse events (AEs) leading to a change in treatment occurred in 10(3%) patients during experimental treatment and in 2(1%) during continuation treatment. All AEs graded as severe and higher, at least possibly related to experimental treatment, finally resolved or improved. Conclusions All arms were shown to be safe and well tolerated, including 35mg/kg of rifampicin. Rifampicin at an increased dose is a promising component for studies of shorter regimens for the treatment of drug‐sensitive TB, with further dose escalation studies warranted. 13 Symposium III – Mycobacteria, Tuberculosis and Malaria ST 14 Characteristics of patients with multidrug‐resistant tuberculosis at the DZIF Clinical Tuberculosis Center (ClinTB), Medical Clinic Borstel I. D. Olaru1,2, J. Heyckendorf1,2, B. Kalsdorf1,2, E. Terhalle1, N. Smitsman1,2, S. Großmann1, C. Lange1,2 1
2
Research Center Borstel, Division of Clinical Infectious Diseases, Borstel, Germany German Center for Infection Research, Clinical Tuberculosis Center Borstel, Borstel, Germany The increase in multidrug‐resistant (MDR) tuberculosis (TB) especially in the WHO region Europe is causing great concern. In Germany the proportion of MDR‐TB among all TB cases is 3.4%, however the number of cases is increasing. The Medical Clinic Borstel hosts the DZIF Clinical Tuberculosis Center (ClinTB), a national referral center for TB treatment, especially MDR‐TB. Objective To evaluate the characteristics of patients with MDR‐TB hospitalized at the DZIF Clinical Tuberculosis Center (ClinTB), Medical Clinic Borstel. Methods Retrospective study of medical records of patients with microbiologically confirmed MDR‐TB hospitalized at the Medical Clinic Borstel 1/2002‐8/2015. Drug susceptibility testing was performed at the National Reference Center for Mycobacteria in Borstel (a WHO supranational reference laboratory). MDR‐TB is defined as resistance to isoniazid and rifampicin, and extensively drug‐resistant (XDR)‐TB as additional resistance to second‐line injectables and fluoroquinolones. Results The study included 73 patients with MDR‐TB with a median age of 39 years, 69% were male. Only 6 (8.2%) patients were German‐born. Two thirds of all patients were born in the former Soviet Union. Sixty‐seven (78%) of were referred from German federal states other than Schleswig Holstein, where the hospital is located. More than half (59%) of patients were admitted since the beginning of 2013, representing a significant increase in admissions. Eight patients (11%) were HIV‐seropositive and 34 (47.2%) received prior TB treatment. Fifty‐
eight (79.5%) patients had pulmonary TB, 5 (6.8%) extrapulmonary TB, and 9 (12.3%) had both pulmonary and extrapulmonary TB. Of the patients with pulmonary TB, 33 (50%) had a positive sputum smear microscopy at MDR‐TB treatment start at the Research Center Borstel. Forty‐three (58.9%) patients had MDR‐TB, 11 (15.1%) had additional resistance to second‐line injectable drugs, 8 (11%) had fluoroquinolone‐resistance (combined 26.1% had pre‐XDR‐TB) and 11 (15.1%) had XDR‐TB. Forty patients (61%) had resistance to all first line drugs. Drug susceptibility patterns are shown in Figure 1. Of the patients with positive sputum cultures on admission, 26 (52%) were culture negative after 8 weeks of therapy, while 44 (88%) were negative after 24 weeks of treatment. Four patients (5%) died during the hospitalization and one had to permanently discontinue therapy due to adverse events. Figure 2 shows the drugs used for treatment. Conclusion Between 2002 and 2015 there is a significant increase in patients with M/XDR‐TB treated at the DZIF ClinTB site, Medical Clinic Borstel. The majority of patients with M/XDR‐TB are foreign‐born and had received prior TB treatment. A large number of patients has pre‐XDR or XDR‐TB, requiring individualized drug management. Figure 1 Figure 2 14 Symposium III – Mycobacteria, Tuberculosis and Malaria ST 15 Host transcriptomics revealing a key to the success story of Mycobacterium tuberculosis U. von Both1,2, M. Berk2, S. M. Newton2, A. Git3, N. Siddiqui2, J. Wright2, S. Hamilton2, M. Levin2 1
Kinder‐und Kinderpoliklinik im Dr. von Hauner'schen Kinderspital, Pädiatrische Infektiologie, Munich, Germany Imperial College, Section of Paediatrics, Paediatric Infection and Immunity, London, Great Britain 3
University of Cambridge, Cancer Research UK Cambridge Institute, CRI, Cambridge, Great Britain 2
Background Mycobacteria survive and multiply inside infected human macrophages by subversion of immune mechanisms critical for intracellular killing and for processing and presentation of mycobacterial antigen for immune recognition by T cells. Although these immune evasion mechanisms are well characterised functionally, the underlying molecular mechanisms are poorly understood. Methods Whole blood from healthy volunteers (BCG‐non‐immunized, tuberculin skin test (TST) negative, healthy individuals) was infected with M. tuberculosis (MTB) and transcriptional genome‐wide profiling was performed. Findings of significantly differentially expressed (SDE) transcripts form a first “discovery” dataset were validated in a second, identically set up, “validation” experiment. Changes in mRNA expression levels were mimicked by protein expression, assessed using FACS and ELISA. Results We found that the predominant genomic response to MTB infection of human blood cells is down‐regulation of genes, with over 70% of the significantly differentially expressed (SDE) genes (log2 FC >1) being down‐
regulated. Spatial, temporal and functional characterisation of repressed genes revealed down‐regulation of multiple genes involved in pathogen sensing and phagocytosis, killing and degradation within the phagolysosome, and processing and presentation of antigen on cell surfaces. Concurrent microRNA profiling identified a set of SDE microRNAs that was significantly associated with the group of SDE mRNAs (enriched for miRNA targets), p=6.9‐31. Conclusion These findings suggest that to survive within infected cells, mycobacteria exploit a mechanism of molecular repression of essential genes within key immunological pathways. MicroRNAs seem to play an important role in this complex gene regulatory off switch event. 15 Symposium III – Mycobacteria, Tuberculosis and Malaria ST 16 The improved and controlled investigation of febrile illness in a rural setting in South‐
Western Tanzania supports the Malaria diagnostic. B. Flach1,2,3, M. Knüpfer4,2,3, C. Mangu5, H. Msila5, H. Schimanowski‐Thomson6, C. Mwansongela6, L. Maboko5 M. Hölscher3,7, N. Heinrich3,7, G. Dobler1,2,3 1
Bundeswehr Institute of Microbiology, Department for Virology and Rickettsiology, Munich, Germany German Partnership Program for Excellence in Biological and Health Security, Munich, Germany 3
German Center for Infection Research, Munich Partner Site, Munich, Germany 4
Bundeswehr Institute of Microbiology, Munich, Germany 5
NIMR‐Mbeya Medical Research Centre, Mbeya, Tanzania 6
Matema Lutheran Hospital, Matema, Tanzania 7
Division for Infectious Diseases and Tropical Medicine, Medical Center of the University of Munich, Munich, Germany 2
Background In Sub‐Saharan Africa febrile illness is one of the major contributions of hospital admissions and death. The WHO recommendation for using malaria rapid diagnostic tests has led to more precise estimates of malaria prevalence in febrile patients than clinical judgement alone. WHO statistics show a decline in malaria‐related admissions and trends from 2000 onwards by more than 50%. We report interims results from a clinical study on febrile disease in outpatients in a rural clinic in Matema, Tanzania, examining the etiology and socio‐
economic covariates of fever of unknown origin. During the study in a GCLP setting the true percentage of Malaria in the patients was tested. Methods Individuals with a body temperature ≥ 37.5 °C, ≥ 1 year of age and with a body weight of ≥ 3.5 kg are being enrolled into the study. Enrollment started in March 2015. Patients are assessed clinically, and receive rapid diagnostic testing (RDT) for malaria following Tanzanian national guidelines. Results 211 patients were tested for malaria, by RDT. 62.1% tested positive, with 45.8% accounted for P. falciparum and 51.9% for P. vivax, P. ovale and P. malariae. 3 patients showed positive bands for both parasites, indicating dual infection. With a positivity rate of 66.7% malaria was found in the age group 12 to < 18 years, followed by 53.9% in patients ≥ 18 years and 51.7% in patients < 12 years of age. Malaria positivity was highest in April and May (71.4% and 71%) and decreased in June (35.5%), parallel to the ending rainy season. The patients reported a median of 3 days (range 1‐30 days) of acute fever at enrollment. Major clinical signs were described by the patients as: Abdominal pain and headache (~50%), followed by vomiting (25,1%), coughing (20.6%), diarrhea and joint pain (10,7%) as well as back pain (7.6%). 13.2 % of all patients enrolled at the clinic presented with a physical exam, a previous malaria test (13.6%) and previous antimalarial treatment (30.2%). Conclusion Despite efforts to control malaria infection in Tanzania, we found high malaria positivity in febrile patients seeking medical attention. The results show the necessity of a controlled test environment, especially in developing areas, to produce high quality results which can be used for proper patient care and treatment. Further analysis of study data is ongoing to identify socio‐economic and environmental risk factors, which would allow targeting intervention to stop disease transmission. 16 Symposium III – Mycobacteria, Tuberculosis and Malaria ST 17 A randomized, controlled, double‐blind, phase 1 clinical trial to evaluate safety, tolerability, immunogenicity and efficacy of CAF01 and aluminum hydroxide as adjuvants for the malaria vaccine candidate GMZ2 in healthy adult African volunteers U. Ateba Ngoa1,2,3, O. P. Nouatin3, J. R. Edoa3, A. A. Adegnika1,2,3, Y. D. Mouwenda3, M. Massinga‐Loembe3 A. L. Bouyoukou Hounkpatin1,3, D. A. Jean Claude3, M. Yazdanbakhsh2, J. Brosnahan1, B. Lell1,3, M. Theisen4 B. Mordmüller1,3, P. Kremsner1,3 1
Institut für Tropenmedizin, Universität Tübingen, Tübingen, Germany Leiden University Medical Center, Department of Parasitology, Leiden, The Netherlands 3
Centre des Recherches Médicales de Lambaréné, Lambaréné, Gabun 4
Statens Serum Institut, Department of Congenetial Disorders, Copenhagen, Denmark 2
GMZ2 is an asexual blood stage candidate that was developed by the Statens Serum Institut, Denmark (SSI) and its partners. It is a fusion protein of conserved fragments of Plasmodium falciparum merozoite surface protein 3 (MSP3) and glutamate rich protein (GLURP). The antigen was selected based on sero‐epidemiological and functional studies and showed excellent safety, tolerability and good immunogenicity when adjuvanted with alum. Results of three phase 1 studies on GMZ2‐alum led to the decision to perform a phase 2 clinical trial in African children to assess safety, tolerability and efficacy in the target population. Results of the trial are still pending but preliminary data support the very good safety and tolerability profile of GMZ2 while efficacy seems modest. In general, data from the four trials suggest that an improved and more durable immune response against the vaccine antigen will result in significantly better protection. Therefore combination of GMZ2 with a novel and more potent adjuvant is the rational next step in clinical development. CAF01 is a novel adjuvant, which was developed by SSI and several clinical trials in Europe and Africa demonstrated good safety, tolerability and improved immunogenicity when combined with various vaccine candidates. This study is performed to compare safety, tolerability, immunogenicity and efficacy of GMZ2 formulated with CAF01 to a control vaccine (Rabies) and 100 μg GMZ2 in alum. A total of 50 volunteers were vaccinated three times by intramuscular injections in the deltoid muscle on alternating sides in four‐week intervals. The participants were divided in 4 groups. 8 Participants received the control vaccine (Group A), 12 participants received 100 μg GMZ2 formulated in alum (Group B), 8 participants received 30 μg GMZ2 formulated in CAF01 (Group C) and 22 participants received 100 μg GMZ2 formulated in CAF01 (Groups D and E). Overall, the vaccination regimen was well tolerated and no serious adverse events nor unsolicited adverse events related to the vaccine occurred. Efficacy of the vaccine will be tested by controlled human malaria infection (CHMI) by direct venous inoculation (DVI) of 3200 PfSPZ Challenge, five to eighteen weeks after completion of the vaccination regimen in September and results will be presented. 17 Symposium IV – Antibiotic resistant bacterial infections: Novel antiinfectives and antibiotic stewardship ST 18 The lectin LecB as target for anti‐infectives against chronic Pseudomonas aeruginosa infections A. Titz1 1
Helmholtz‐Institute for Pharmaceutical Research Saarland, Chemical Biology of Carbohydrates, Saarbrücken, Germany The opportunistic pathogen Pseudomonas aeruginosa is currently considered as a major threat to public health care. The Gram‐negative bacterium forms biofilms which protects itself from host defense and antibiotic therapy resulting in chronic infections. The bacterial lectin LecB is a virulence factor and plays a prominent role in biofilm formation. Inhibition of lectin function with carbohydrate‐based ligands was shown to disrupt bacterial biofilm formation. Based on the crystal structure of the lectin with its glycan ligands, we dissected the contributions of individual functional groups to protein binding in a biophysics‐guided approach. This knowledge was then used for the development of small and drug‐like glycan‐based molecules as LecB inhibitors as future anti‐biofilm compounds in chronic Pseudomonas aeruginosa infections. 1. Sommer, R.; Hauck, D.; Varrot, A.; Wagner, S.; Prestel, A.; Möller, H.M.; Imberty, A.; Titz, A. ChemistryOpen 2015, doi: 10.1002/open.201500162. 2. Hofmann, A.; Sommer, R.; Hauck, D.; Stifel, J.; Göttker‐Schnetmann, I.; Titz, A. Carbohydr. Res. 2015, 412, 34‐42. 3. Sommer, R.; Exner, T.E.; Titz, A. PLoS ONE 2014, 9(11): e112822. 4. Hauck, D.; Joachim, I.; Frommeyer, B.; Varrot, A.; Philipp, B.; Möller, H.M.; Imberty, A.; Exner, T.E.; Titz, A. ACS Chem. Biol. 2013, 8(8), 1775–1784. 18 Symposium IV – Antibiotic resistant bacterial infections: Novel antiinfectives and antibiotic stewardship ST 19 Development of a virulence gene scoring system for ESBL‐producing E. coli isolates from human and animal sources J. Schmiedel1, H. Ghosh1, A. Höland2, L. Falgenhauer1, C. Imirzalioglu1, T. Chakraborty1 1
2
Medizinische Mikrobiologie, Forschung, Giessen, Germany Medizinische Informatik, Giessen, Germany Extended‐spectrum β‐lactamase (ESBL)‐producing multidrug‐resistant Escherichia (E.) coli are an increasing problem in human and veterinary medicine. Many studies address the resistance characteristics of ESBL‐
producers. However, only few studies have been carried out invesigating their virulence properties. This study focused on virulence properties enabling sepsis formation and the development of an in‐silico based virulence gene scoring system for ESBL‐producing E. coli. For this purpose the virulence and resistance gene content of 101 selected ESBL‐positive E. coli isolates from human and animal sources was investigated. All strains were submitted to whole genome sequencing and screened for the presence of virulence and resistance genes. Genes that can be used as virulence markers for sepsis development were identified. 40 E. coli isolates (human isolates = 20, animal isolates = 20) were tested in the Galleria (G.) mellonella model for in vivo virulence.Genotypic and phenotypic data was correlated with the cox regression to identify virulence gene markers enhancing sepsis development. Genes and operons from the categories iron metabolism, serum resistance and adhesins were found in almost all isolates. Isolates from the phylogenetic group B2 and D contained considerably more virulence factors in comparison to other phylogenetic groups. Potential virulence markers were identified based on the presence of genes and gene combinations enabling serum resistance. These include iron acquisition genes and genes necessary for overcoming host defense mechanisms. The operons coding for aerobactin, yersiniabactin, salmochelin and sit as well as the genes hlyF, iss, ompT, malX, the toxin genes astA, set1AB, senB and genes encoding the capsule gene cluster were selected as potential virulence markers. Isolates containing the aforementioned genes and combinations of iss/sit‐operon/siderophore (aerobactin, yersiniabactin, salmochelin) were selected for in vivo virulence testing in G. mellonella. More than half of the investigated isolates (56 %) were found to be clearly larvicidal as defined by larval killing within 24 hours. Statistical analysis using the cox regression identified the absolute number of virulence genes present in the isolates, affiliation to ST131 and ST410, malX, hlyF, the combinations iss/Aerobactin, sitA/Aerobactin, iss/Yersiniabactin as risk factors for high larvicidal effects in G. mellonella. The identified genes and gene combination are promising candidates to establish a genome based scoring system for ESBL‐producing E. coli. Future studies will expand on these findings and serve to refine the preliminary scoring system. 19 Symposium IV – Antibiotic resistant bacterial infections: Novel antiinfectives and antibiotic stewardship ST 20 Quality assurance in blood culture diagnostics ‐ the Thuringian prospective population‐
based registry AlertsNet A. Karch1,2, R. P. Schmitz3, F. Rißner3, M. Jakob3, R. T. Mikolajczyk1,2, F. M. Brunkhorst3 1
German Centre for Infection Research, Hannover‐Braunschweig site, Braunschweig, Germany Helmholtz Centre for Infection Research, ESME ‐ Epidemiological and Statistical Methods Research Group, Braunschweig, Germany 3
Center for Clinical Studies Jena (ZKS), Center for Sepsis Control and Care (CSCC), Jena University Hospital, Jena, Germany 2
Introduction Blood culture (BC) testing before initiation of antimicrobial therapy is recommended as a standard of care in international sepsis guidelines and has been shown to reduce intensive care unit stay, antibiotic use, and costs in hospitalized patients. A recently established population‐based study for the German Federal state of Thuringia started in 2014 and connects hospitals and microbiological laboratories within an electronic registry for immediate integration and evaluation of BC findings (AlertsNet, www.alertsnet.de). Methods Data from seven hospitals with five associated labs were included in this analysis. Microbiological data of all BCs taken in the participating hospitals as well as clinical data from all patients with clinically relevant positive BCs collected from May 2014 to May 2015 and were analyzed using standard measures of descriptive statistics. Results In total, 28,309 BC sets have been taken in the participating hospitals. After excluding negative BCs as well as contaminants and after merging positive BCs of the same patient taken within 96 hours, a total of 1,882 clinically relevant BC sets were identified representing 812 patients (60.0% male) with a median age of 71 (IQR: 59‐79) years. Of these, 60.8% suffered from a nosocomial bloodstream infection, while the remaining 39.2% were infected outside the hospital setting. Disease progression in the 96 hours after BC sampling was heterogeneous and ranged from sepsis without organ dysfunction (50.8%) to septic shock (22.2%). The overall case fatality rate was 23.3%. Risk factors for death include the need for central lines (OR 3.10, 95% 2.24‐4.30), mechanical ventilation (OR 6.73, 95% 4.56‐9.93) and pneumonia being the primary focus of the bloodstream infection (OR 1.88, 95% 1.31‐2.69). The most common pathogens were Escherichia coli (19.7%), Staphylococcus aureus (16.5%) and Staphylococcus epidermidis (13.6%). Pneumonia (18.8%) and urinary tract infections (17.0%) were the most common foci in the study population; in 35.2% of patients, no focus could be detected. Conclusions Within the first 12 months of follow up, AlertsNet provides data on BC positive patients with a wide clinical range of bloodstream infections. Risk factors for a fatal disease outcome known from other studies could be replicated underlining the internal validity of AlertsNet. Distribution of pathogens and underlying foci resembled the experience of previous population‐based studies in other countries. 20 Symposium IV – Antibiotic resistant bacterial infections: Novel antiinfectives and antibiotic stewardship ST 21 Molecular characterization of third generation cephalosporin‐resistant Enterobacteriaceae (3GCREB) on hospital admission ‐ results from the first ATHOS prevalence study A. Hamprecht1, A. M. Rohde2, S. Feihl3, A. Mischnik4, B. Obermann5, S. Peter6, F. Schwab2, T. Wille1, J. Zweigner1,2 W. Kern4, P. Gastmeier2, H. Seifert1 1
Uniklinik Köln, Institut für medizinische Mikrobiologie, Immunologie und Hygiene, Cologne, Germany Charité Universitätsmedizin, Institut für Hygiene und Umweltmedizin, Berlin, Germany 3
Technische Universität München, Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Munich, Germany 4
Universitätsklinikum Freiburg, Abteilung Infektiologie, Freiburg, Germany 5
Universitätsklinikum Schleswig‐Holstein, Institut für Medizinische Mikrobiologie und Hygiene, Lübeck, Germany 6
Universitätsklinikum Tübingen, Institut für Medizinische Mikrobiologie und Hygiene, Tübingen, Germany 2
Introduction The ATHOS study (antibiotic therapy optimisation study) is a multicentre study which aims at collecting prevalence and incidence data for carriage of multi‐drug resistant organisms (MDROs) and to implement antibiotic stewardship programs in the inpatient and outpatient setting. As part of this project, the first prevalence study was carried out in 2014 at six German university hospitals. Objectives To assess the colonization of patients with third generation cephalosporin‐resistant Enterobacteriaceae (3GCREB) on hospital admission and to characterize the resistance mechanisms of 3GCREB. Methods Adult patients were screened for 3GCREB carriage in six German university hospitals in 2014. Rectal swabs or stool samples were taken from patients within 72 h of admission. Isolates with 3GCREB were characterized for the presence of extended spectrum beta lactamases (ESBL), AmpC, and carbapenemases by phenotypic and molecular methods. Results A total of 4376 patients were included in the prevalence survey. Of these, 417 patients were 3GCREB carriers (9.5% prevalence). Of 439 isolates, 414 (94.3%) were available for molecular and phenotypic analysis of underlying resistance mechanisms to third generation cephalosporins. Escherichia coli was the most frequent 3GCREB organism detected at all six institutions, with 339 of 414 isolates (81.9%), followed by Klebsiella pneumoniae (N=36, 8.7%), Enterobacter spp. (N=19, 4.6%) and Citrobacter spp. (N=14, 3.4%). ESBL was the dominant resistance mechanism and detected in 375/414 isolates (90.6%), with most isolates belonging to CTX‐
M‐1 group (277/414; 66.9%). Of the CTX‐M‐1 group, CTX‐M‐15 and CTX‐M‐1 were the most frequent ESBL types. ESBLs of the CTX‐M‐9 group were detected in 70 isolates (16.9%), SHV‐ESBLs in 23 isolates (5.6%) and AmpC producing Enterobacteriaceae in 41 isolates (9.9%). Carbapenemase producing Enterobacteriaceae were still rare (N=5, 1.1%; prevalence, 0.11%), with VIM‐1 being the most frequent carbapenemase (N=3). Conclusion To our knowledge, this is the largest study on the prevalence of 3GCREB carriage among hospitalized patients in Germany. The prevalence of 3GCREB carriage on admission was 9.5% and higher as previously reported. E. coli was the most common 3GCREB species, with resistance mediated usually by ESBLs (most commonly of CTX‐
M‐1 group). Carbapenemases were still rare with VIM‐1 being the most common carbapenemase detected in our population. 21 Symposium IV – Antibiotic resistant bacterial infections: Novel antiinfectives and antibiotic stewardship ST 22 Characterization and quantification of antimicrobial resistance selection pressure caused by antibiotic treatment: a sequence based approach M. Willmann1,2, M. El‐Hadidi3, D. Huson3, M. Schütz1,2, C. Weidenmaier1,2, I. Autenrieth1,2, S. Peter1,2 1
University of Tübingen, Institute of Medical Microbiology and Hygiene, Tübingen, Germany German Center for Infection Research (DZIF), partner site Tübingen, Tübingen, Germany 3
University of Tübingen, Center for Bioinformatics, Tübingen, Germany 2
The human gut is densely populated by a large and diverse number of bacteria termed the gut microbiota. Antibiotic treatment has a significant impact on the gut microbiota and may lead to the selection of antimicrobial resistance genes (ARGs) in the human intestine. In order to determine the selection pressure and changes of ARGs we have followed two healthy volunteers receiving a six‐days course of ciprofloxacin (Cp) treatment. By applying ultra deep metagenome shotgun sequencing in combination with different bioinformatical tools, we established a pipeline to identify and quantify ARGs in the humane intestine. Fixed‐ and random‐effects models were used to uncover the change in ARG abundance per defined daily dose of Cp as an expression of the respective selection pressure. Antibiotic perturbation led to a change in the microbiota composition in both individuals, with an incomplete reconstitution at day 28 after treatment. For the determination of the changes of ARG abundance in the human gut resistome three different quantification methods were established and compared to each other. Various shifts in the ARG abundance were observed in both volunteers with a trend toward a negative selection for class A beta‐lactamases (‐2.66 hits per million sample reads / defined daily dose; p = 0.06). In one individual a strong positive selection for class D beta‐lactamases was detected. One of the class D beta‐lactamases was located on a mobile genetic element. By 4 weeks after the end of treatment, the resistome composition returned toward the initial state but to a different degree in both individuals. We have successfully established an analysis pipeline for the identification and quantification of AGRs in the human gut intestine to determine the selection pressure of antimicrobial agents. This promising approach can be applied in clinical settings to compare therapeutic regimes regarding their effect on the intestinal resistome. Choosing antibiotics with a low potential to select for clinically relevant resistance genes might result in a decreased spread of resistance genes and thereby reduce the burden of infections with multi‐drug resistant pathogens. 22 Symposium IV – Antibiotic resistant bacterial infections: Novel antiinfectives and antibiotic stewardship ST 23 Isolation and characterization of antibacterial cyclophanes and related analogs from cyanobacteria of the genera Nostoc and Cylindrospermum M. Preisitsch1, T. Niedermeyer2,3, K. Harmrolfs4,5, M. Schneefeld6, J. Herrmann5,7, C. Wiesner8, F. Bange6 R. Müller 5,7, S. Mundt1 1
Institut für Pharmazie, Pharmazeutische Biologie, Greifswald, Germany Eberhard Karls Universität Tübingen, Tübingen, Germany 3
Deutsches Zentrum für Infektionsforschung, Standort Tübingen, Tübingen, Germany 4
Universität des Saarlandes, Pharmazeutische Biotechnologie, Saarbrücken, Germany 5
Helmholtz‐Institut für Pharmazeutische Forschung Saarland, Saarbrücken, Germany 6
Medizinische Hochschule Hannover, Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Hannover, Germany 7
Deutsches Zentrum für Infektionsforschung, Standort Hannover‐Braunschweig, Hannover‐Braunschweig, Germany 8
Sealife PHARMA GmbH, Tulln, Austria 2
A screening of cyanobacteria extracts revealed an extract from a Nostoc sp. as highly active against Gram‐
positive pathogens. The active compounds were isolated and structurally characterized, resulting in 5 new and 11 known cyclophanes. In addition to a pronounced antibacterial activity against methicillin‐resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae (MIC 0.1‐2 µM), the compounds displayed considerable cytotoxicity profiles, with IC50 values of 3‐12 µM against HaCaT cells. Presence of a carbamoyl group slightly enhances both antibacterial and cytotoxic activity. After development of a rapid and exhaustive one‐step extraction protocol, a screening of > 100 cyanobacteria strains for novel analogs was conducted. This led to the isolation of the cylindrofridines, novel “ring‐opened” cyclophane analogs. Interestingly, the antibacterial activity of the two cyclophane‐like congeners is much lower (MIC > 75 µM), while their cytotoxicity is comparable (IC50 25 µM). In contrast, the monomeric compound still displays antibacterial activity (MIC 8‐17 µM), but lower cytotoxicity (IC50 100 µM), showing that antibacterial and cytotoxic activity of this compound class can potentially be unlinked. Presence of bromide in the cultivation medium led to the biosynthesis of brominated cyclophanes. Comparative activity testing showed that bromine substituting chlorine has a neglectable effect on bioactivity against MRSA and S. pneumoniae but revealed significant differences in anti‐Mycobacterium tuberculosis activity of carbamoylated cyclophanes. 23 Symposium V – Chronic Viral Infections: HIV and Hepatitis ST 24 Targeted cleavage of the HIV‐1 proviral genome using AAV Vector‐Mediated CRISPR M. Nickl1,2, K. Börner1,2, F. Schmidt1, A.‐ C. Gnirck1, P. Bayer1, J. Meichsner1, N. Kurzawa1, V. Laketa1,2 D. Grimm1,2, H.‐ G. Kräusslich1,2 1
2
University Hospital Heidelberg, Department of Infectious Diseases, Virology, Heidelberg, Germany German Center for Infection Research (DZIF), partner site Heidelberg, Heidelberg, Germany The current highly active antiretroviral therapy significantly decreases the viral burden in HIV‐infected patients, but virus eradication and cure cannot be achieved due to latent viral reservoirs. Accordingly, treatment interruption generally leads to a rapid rebound of viremia, mainly from latently infected long‐living CD4+ T‐cells. This project aims at targeting and ablating integrated HIV‐1 proviral genomes in infected cells by CRISPR/Cas delivered via adeno‐associated viral (AAV) vectors. Therefore, we first screened AAVs with peptide insertions and identified novel peptide‐capsid combinations that mediate 100% transduction efficiency of human T‐cell lines. In parallel g(uide)RNAs (that attract the Cas9 to a specific DNA sequence) were engineered to target either the viral long terminal repeats or protein‐coding sequences within the HIV‐1 genome. Using a T7 endonuclease assay we showed successful cleavage of the viral genome in a HeLa‐derived cell line carrying a stably integrated HIV‐1 genome and in J‐Lat cells, a commonly used model of HIV latency. DNA sequencing revealed mutations at desired HIV‐1 target sites in both cell lines. To further increase the probability of destroying the proviral genome, vectors co‐expressing three gRNAs under different promoters were designed to simultaneously cleave three separate target sites in HIV‐1. Results from T7 endonuclease assays confirmed successful concurrent cleavage of these target sites in J‐Lat cells, and FACS analysis revealed a 35% decrease in proviral expression. Moreover, an ~90% reduction of HIV‐1 infectivity was observed when HeLa‐P4 cells were pre‐treated with the AAV CRISPR/Cas multiplexing vectors prior to infection with HIV‐1. Collectively, our results demonstrate that combinatorial AAV/CRISPR strategies can effectively cleave the integrated HIV‐1 genome, reduce HIV‐1 gene expression, and provide a protective effect against HIV, highlighting their potential to inactivate and/or purge the latent HIV‐1 reservoir. 24 Symposium V – Chronic Viral Infections: HIV and Hepatitis ST 25 High‐efficiency knockout of HIV co‐receptor CCR5 in primary T cells after mRNA transfection of the novel transcription activator‐like effector nuclease CCR5‐Uco‐TALEN U. Mock1, R. Machowicz1, I. Hauber2, S. Horn1, P. Abramowski1, B. Berdien1, J. Hauber2,3, B. Fehse1 1
UK Hamburg‐Eppendorf, Forschungsabt. Zell‐ und Gentherapie, Hamburg, Germany Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany 3
German Center for Infection Research (DZIF), partner site Hamburg, Hamburg, Germany 2
Besides its physiological role, the chemokine receptor CCR5 plays an essential part during HIV infection, acting as the co‐receptor for so‐called R5‐tropic strains that usually mediate initial infection. App. 1% of Caucasians are homozygous for a natural deletion variant of CCR5 (CCR5Δ32) and, based thereon, resistant towards infection with R5‐tropic HIV strains. The “Berlin patient” provided evidence that resistance can be acquired even in case of established HIV infection by transfusion of CCR5‐negative cells. As a consequence, CCR5 has become an interesting target for gene‐editing strategies using designer nucleases. We have recently introduced a new transcription activator‐like effector nuclease, CCR5‐Uco‐TALEN for highly specific CCR5 knockout. Long‐
term and/or high‐level expression of designer nucleases increases the risk of unwanted off‐target activity. Therefore, we established an expedient mRNA‐electroporation protocol for primary T lymphocytes ‐ a gentle and truly transient method for efficient TALEN delivery. By means of this technique we were able to regularly obtain high‐rate CCR5 knockout (> 90% in PM1 and >50 % in primary T cells) combined with low off‐target activity, as demonstrated by flow cytometry and next‐generation sequencing. We also devised and validated a novel, very handy digital‐PCR technique that facilitates easy and fast detection of gene‐knockout frequencies with a sensitivity of at least 0.2%. Using functional assays we showed that CCR5‐edited cells were not only resistant towards HIV‐derived lentiviral vectors, but also protected from infection with the replication‐
competent CCR5‐tropic HIV‐1BaL strain. Indeed, exposure to HIV‐1BaL resulted in a profound selective advantage of CCR5‐gene edited T cells in vitro and, in four of five humanized mice, in vivo. In summary, we have introduced a novel TALEN for high‐efficiency knockout of CCR5. Our mRNA‐based gene‐editing protocol does not rely on viral vectors and should therefore be highly compatible with large‐scale production and good‐
manufacturing practice. We therefore suppose that CCR5‐Uco‐TALEN has a great potential for clinical translation. 25 S y m p o si u m
V – C h ro ni c V i ral I nf ec tio ns: H I V and epatitis
S T 2 6
E ng i neeri ng and anal y si s o f B rec 1: A B ro ad- rang e rec o m b i nase th at speci cally targ ets th e
m aj o ri ty o f H I V - 1 i so l ates
J . Hauber1, J . Karpinski1,2, I. Hauber1, J . C hemnitz1, C . Schä fer1, N. Beschorner1, J . van Lunzen3, F. Buchholz2
1
Heinrich P ette Institute - Leibniz Institute for Experimental Virology, Hamburg, Germany
TU Dresden, Dresden, Germany
3
University Medical C enter Hamburg-Eppendorf, Hamburg, Germany
2
C urrent drugs against HIV cannot clear the patient from the virus. Therefore, finding a cure for HIV infection
remains a high priority of HIV/ AIDS research. We recently generated a recombinase (termed Tre) tailored to
e ciently eradicate the provirus from the host genome of HIV-1 infected cells by specifically targeting a
sequence that is present in the long terminal repeats (LTRs) of the viral DNA. I n v iv o analyses in HIV-infected
humanized mice demonstrated highly significant antiviral effects of Tre-recombinase. However, the fact that
Tre recognises a particular HIV-1 subtype A strain may limit its broad therapeutic application. To advance our
Tre-based strategy toward a universally e cient cure, we have now engineered broad-range recombinase 1
(Brec1), a recombinase applicable to the maj ority of HIV-1 infections by the various virus strains and subtypes.
Brec1 removes e ciently, precisely and safely the integrated provirus from infected cells. Moreover, Brec1 acts
e caciously on clinical HIV-1 isolates in v it ro and in v iv o , particularly in humanized mice that were
“ personalized” with patient-derived cells. Thus, Brec1 represents a novel and potent antiviral reagent
per i ng broad clinical application in the context of future curative HIV-1 therapy strategies.
Zur Behandlung von HIV-Infektionen
DOLUTEGRAVIR-BASIERTE REGIME*
Starker Start. Starke Basis. Für Ihre Patienten.
dolutegravir/abacavir/
lamivudin
Einfach: Das onepill-Regime
mit Dolutegravir
* DTG + Nukleosidanaloga gemäß der DAIG- und EACS Leitlinien
Flexibel: Für maßgeschneiderte
Therapie-Regime
www.dolutegravir.com
Tivicay® 50 mg Filmtabletten; Triumeq® 50 mg/600 mg/300 mg Filmtabletten
Wirkstoffe: Tivicay Dolutegravir; Triumeq Dolutegravir/ Abacavir/ Lamivudin, Zusammensetzung: Jede Filmtablette Tivicay enthält Dolutegravir-Natrium, entsprechend 50 mg Dolutegravir; Jede Filmtablette Triumeq enthält 50 mg Dolutegravir
(als Natrium-Salz), 600 mg Abacavir (als Sulfat) und 300 mg Lamivudin. Sonstige Bestandteile: Tivicay: Mannitol (E421), mirokristalline Cellulose, Povidon K29/32, Poly(O-carboxymethyl)stärke-Natriumsalz, Natriumstearylfumarat, Poly(vinylalkohol),
Titandioxid (E171), Macrogol, Talkum und Eisen(III)-hydroxid-oxid (E172), Triumeq: Mannitol (E421), mikrokristalline Cellulose, Povidon K29/32, Poly(O-carboxymethyl)stärke-Natriumsalz, Magnesiumstearat, Opadry II Violett 85F90057 (enthält
Poly(vinylalkohol), Titandioxid, Macrogol, Talkum, Eisen(II,III)-oxid, Eisen(III)-oxid). Anwendungsgebiete: Tivicay in Kombination mit anderen antiretroviralen Arzneimitteln zur Behandlung von Infektionen mit dem humanen Immundefizienz-Virus (HIV)
bei Erwachsenen und bei Jugendlichen im Alter von über 12 Jahren. Triumeq zur Behandlung von HIV-Infektionen bei Erwachsenen und Jugendlichen über 12 Jahren, die mindestens 40 kg wiegen. Vor Beginn der Behandlung mit Abacavir-haltigen
Arzneimitteln sollte jeder HIV-infizierte Patient auf das Vorhandensein des HLA-B*5701-Allels hin untersucht werden. Patienten, bei denen bekannt ist, dass sie das HLA-B*5701-Allel tragen, sollten Abacavir nicht anwenden. Gegenanzeigen: Tivicay
und Triumeq: Überempfindlichkeit gegen einen der Wirkstoffe oder einen der sonstigen Bestandteile. Einnahme von Dofetilid. Nebenwirkungen: Tivicay: Sehr häufig: Kopfschmerzen, Durchfall, Übelkeit. Häufig: Hautausschlag, Juckreiz, Erbrechen,
Magenschmerzen (Bauchschmerzen), Magenbeschwerden (Beschwerden im Bauchraum), Schlafstörungen, Schwindel, Anormale Träume, Depression, Erschöpfung, Blähungen, Anstieg der Leberenzymwerte im Blut, Anstieg der Kreatin-Phosphokinase.
Gelegentlich: Allergische Reaktionen, Hepatitis, Suizidgedanken oder suizidales Verhalten (insbesondere bei Patienten, die zuvor eine Depression oder psychische Erkrankung hatten);Triumeq: Sehr häufig: Kopfschmerzen, Durchfall, Übelkeit,
Schlafstörungen, Fatigue. Häufig: Überempfindlichkeitsreaktion, Appetitlosigkeit, Hautausschlag, Juckreiz, Erbrechen, Magenschmerzen (Bauchschmerzen), Magenbeschwerden (Beschwerden im Bauchraum), Verdauungsstörungen, Blähungen,
Schwindel, Anormale Träume, Albträume, Depression, Müdigkeit, Fieber, Husten, Gereizte und laufende Nase, Haarausfall, Muskelschmerzen und -beschwerden, Gelenkschmerzen, Schwächegefühl, Allgemeines Unwohlsein, Anstieg der Leberenzymwerte,
Anstieg der Kreatinin-Phosphokinase. Gelegentlich: Leberentzündung, Thrombozytopenie, Anämie, Neutropenie, Hyperglykämie, Hyperlipidämie, Suizidgedanke oder Suizidversuch (insbesondere bei Patienten mit vorbestehender Depression oder
psychischer Erkrankung). Selten: Pankreatitis, Rhabdomyolyse, Anstieg der Amylase. Sehr selten: Taubheit, kribbelndes Gefühl an der Haut, Schwächegefühl in den Gliedmaßen, Erythema multiforme, Stevens-Johnson-Syndrom, toxische epidermale
Nekrolyse, aplastische Anämie. Weitere Nebenwirkungen: Tivicay und Triumeq: klinisch nicht relevante Erhöhung der Serum-Kreatininwerte innerhalb der ersten Behandlungswoche, die über 48 Wochen stabil blieben. Mögliche Nebenwirkungen
einer antiretroviralen Kombinationstherapie: Immun- Rekonstitutions -Syndrom, Osteonekrose, opportunistische Infektionen. Warnhinweis Triumeq: Patientenpass: „ACHTUNG! Bei Verdacht auf eine Überempfindlichkeitsreaktion wenden Sie sich
SOFORT an Ihren Arzt. Entnehmen Sie die beiliegende Warnhinweiskarte, diese enthält wichtige Sicherheitsinformationen.” Verschreibungspflichtig. Stand: Tivicay® Juli 2015, Triumeq® Juni 2015/1. ViiV Healthcare GmbH, 80700 München.
www.viivhealthcare.com
Weitere Informationen über Tivicay:
Dosierung und Art der Anwendung: 50 mg einmal täglich bei Erwachsenen und Jugendlichen ohne Integrase-Inhibitor-Resistenz und mindestens 40 kg Körpergewicht. In Kombination mit Enzym-induzierenden Mitteln, Etravirin (ohne geboosterte
Protease-Inhibitoren), Tipranavir/Ritonavir, Rifampicin, Johanniskraut, Efavirenz, Nevirapin oder bestimmten Antiepileptika 50 mg zweimal täglich. 50 mg zweimal täglich bei Erwachsenen mit Integrase-Inhibitor-Resistenz (dokumentiert oder klinisch
vermutet) und mindestens 40 kg Körpergewicht, bevorzugt mit einer Mahlzeit. Bei Patienten mit Integrase-Inhibitor-resistenten Viren und eingeschränkten Behandlungsoptionen aufgrund fortgeschrittener Mehrklassen-Resistenz könnte die Wirksamkeit
durch zweimal täglich 100 mg erhöht werden (hohe Dosis nicht in Kombination mit Atazanavir). Dosisanpassungen bei Komedikation siehe Fachinformation. Weitere Warnhinweise und Vorsichtsmaßnahmen laut Fachinformation: Anpassung der
Metformin-Dosis bei Beginn und Beendigung der gleichzeitigen Anwendung bzw. eine Reduktion der Metformin-Dosis unbedingt bei Patienten mit eingeschränkter Nierenfunktion erwägen; Vorsicht bei schwerer Leberfunktionsstörung (Child-Pugh-Grad
C). Bei Integrase-Inhibitor-Resistenz: gleichzeitige Anwendung von Arzneimitteln, die die Dolutegravir-Exposition reduzieren sollte vermieden werden (z. B. magnesium- oder aluminiumhaltige Antazida, eisen- und calciumhaltige Ergänzungsmittel,
Multivitaminpräparate und Enzym-induzierende Mittel).. Weitere Informationen siehe Fachinformation.
Weitere Informationen über Triumeq:
Dosierung und Art der Anwendung: Eine Tablette einmal täglich bei Erwachsenen und Jugendlichen ab 12 Jahren, die mindestens 40 kg wiegen; kann mit oder ohne eine Mahlzeit eingenommen werden. Triumeq ist eine fixe Kombination und
darf nicht für Patienten verschrieben werden, die eine Dosisanpassung eines der Bestandteile benötigen. Monopräparate mit Dolutegravir, Abacavir und Lamivudin stehen zur Verfügung. Weitere Warnhinweise und Vorsichtsmaßnahmen laut
Fachinformation: Vorsicht bei gleichzeitiger Anwendung von Abacavir mit Ribavirin, bei Patienten mit mäßiger oder schwerer Lebererkrankung (inkl. HBV und HCV). Gleichzeitige Anwendung nicht empfohlen: Lamivudin, Emtricitabin und Cladribin
sowie Arzneimittel, die die Dolutegravir-Exposition reduzieren. Anpassung der Metformin-Dosis bei Beginn und Beendigung der gleichzeitigen Anwendung bzw. eine Reduktion der Metformin-Dosis unbedingt bei Patienten mit eingeschränkter
Nierenfunktion erwägen. Eine kausale Beziehung zwischen der Behandlung mit Abacavir und dem Risiko für einen Myokardinfarkt kann derzeit weder bestätigt noch widerlegt werden. Hypersensitivitätsreaktion: Sowohl Abacavir
als auch Dolutegravir sind mit dem Risiko einer Überempfindlichkeitsreaktion assoziiert. Es ist keine klinische Differenzierung der verursachenden Substanz möglich. Schwere Überempfindlichkeitsreaktionen wurden häufiger
mit Abacavir beobachtet, assoziiert mit positivem Test auf HLA-B*5701-Allel; auch ohne HLA-B*5701 ist eine Reaktion möglich; vor Beginn der Behandlung mit Abacavir sollte auf HLA-B*5701 getestet werden; HLA-B*5701 Träger
sollten Triumeq nicht anwenden. Bei vermuteter Überempfindlichkeitsreaktion muss Triumeq sofort abgesetzt werden und darf (ebenso wie andere Arzneimittel, die Abacavir oder Dolutegravir enthalten) nie wieder eingenommen
werden. Weitere Informationen siehe Fachinformation.
Diese Arzneimittel unterliegen einer zusätzlichen Überwachung. Dies ermöglicht eine schnelle Identifizierung neuer Erkenntnisse über die Sicherheit. Angehörige von Gesundheitsberufen sind aufgefordert, jeden
Verdachtsfall einer Nebenwirkung dem Bundesinstitut für Arzneimittel und Medizinprodukte, Abt. Pharmakovigilanz, Kurt-Georg-Kiesinger-Allee 3, D-53175 Bonn, Website: www.bfarm.de zu melden.
DE/DGR/0005/15(1)a 09.2016
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Symposium V – Chronic Viral Infections: HIV and Hepatitis ST 27 Differential European trends in HBV prevalence over the last decades J. Ott1,2,3, J. Horn3, R. T. Mikolajczyk1,2,3 1
German Centre for Infection Research (DZIF), Hannover‐Braunschweig Site, Braunschweig, Germany Hanover Medical School (MHH), Hanover, Germany 3
Helmholtz‐Centre for Infection Research, Dept. of Epidemiology, Braunschweig, Germany 2
Introduction Availability of data on chronic HBV infection seroprevalence (as measured by HBsAg) in the general population shows great variation according to country, with those regions of higher HBV endemicity, e.g. Eastern European states having fewer data and more periods without any data. No epidemiologic assessment has been conducted on country‐specific changes in HBV prevalence over the last decades in order to identify time patterns across European countries. Objective Based on seroprevalence estimates of chronic HBV infection generated applying a generalized linear model to data published between 1965 and 2013, the objective is to identify time trends of chronic HBV infection and corresponding patterns in Europe. Results Among 1,910 studies identified on HBsAg seroprevalence worldwide, 1,009 were conducted in Europe, encompassing 52,154,308 study subjects. Continuously low prevalence of chronic HBV infection with a slow and significant declining trend since 1970 is shown for Western European countries like France, Germany, Spain, UK, and The Netherlands. Eastern European countries such as the Russian Federation, Romania and Georgia show stable pattern of HBV prevalence from 1980 to 2007. Albania, a country of highest HBV prevalence in Europe (overall 7.8%), demonstrates the strongest decline in chronic HBV prevalence over the last decades. To a similar extent, HBV endemicity also declined in Greece (from 4.7% to 0.5%), Turkey (from 9.3% to 3.1%), and Italy (from 2.7% to 1.1%). Conclusion In the context of population developments such as population aging and movement, results have implications for both, reconsidering preventive measures in place, e.g. HBV vaccination policies, and for diagnostic and therapeutic investments. This is further underscored by the differential time patterns within Europe, e.g. the significant decreases in South‐Eastern Europe and the stable, relatively high HBV prevalence in some Eastern European countries. Changes are likely to be associated with onset and coverage of HBV vaccination programs. 27 Symposium V – Chronic Viral Infections: HIV and Hepatitis ST 28 Suppression of HBV by RNAi restores HBV‐specific immunity and enhances the efficacy of therapeutic vaccination A. Kosinska1, T. Michler2, C. Jäger2, N. Röder1, U. Protzer1,2 1
2
Helmholtz Zentrum München, Institut für Virologie, Munich, Germany Technische Universität München, Institut für Virologie, Munich, Germany More than 240 million people worldwide are chronically infected with hepatitis B virus (HBV) and at risk of developing liver cirrhosis and hepatocellular carcinoma. Numerous studies showed that HBV persistence correlates with a failure to develop an efficient virus‐specific T‐cell response. Thus, induction of HBV‐specific immune responses by therapeutic vaccination may be a promising strategy to treat chronic hepatitis B. However, such strategies have shown only limited efficacy so far. It is assumed that high levels of circulating viral antigens during chronic infection may induce HBV tolerance. We hypothesized that suppression of HBV proteins via RNAi could help to restore HBV‐specific immunity and enhance the efficacy of therapeutic vaccination. To achieve this, we treated HBV transgenic mice with an adeno‐associated virus serotype 8 (AAV8) encoding for a short‐hairpin RNA (shRNA), which targets the HBV‐
transcripts (AAV‐shHBV). A vector expressing an irrelevant shRNA served as control (AAV‐shNEG). Eight weeks later, mice received protein prime ‐ modified vaccinia Ankara (MVA) boost therapeutic immunization with HBV core and surface antigens (HBcAg and HBsAg) or a control vaccination. Intravenous injection of AAV‐shHBV resulted in up to 99% reduction of serum HBsAg and HBeAg levels, which was not seen after AAV‐shNEG treatment. Moreover, mice which had received either (1) AAV‐shNEG and the HBV vaccine, or (2) AAV‐shHBV and the control vaccine did not elicit any or only weak HBV‐specific CD8 T‐cell responses. In contrast, all mice treated with AAV‐shHBV and the HBV vaccine developed HBs‐, as well as HBc‐specific CD8 T‐cell responses in the liver, which were significantly higher (HBs p<0.01; HBc p<0.05) then in mice treated with AAV‐shNEG and the HBV vaccine. Interestingly, HBs‐specific CD8 T‐cell responses in the liver correlated inversely with HBeAg (p<0.001) as well as HBsAg (p<0.01) levels at week 8 when the vaccination scheme was started. Mice treated with AAV‐shHBV and the HBV vaccine also showed a rise of ALT activity after vaccination, which correlated significantly (p<0.05) with HBs‐specific CD8 T‐cell responses in the liver. In summary, these results show that circulating HBV proteins prevent the induction of HBV specific T‐cell responses. RNAi‐mediated suppression of HBV transcripts prior to therapeutic vaccination facilitates the induction of HBV‐specific T‐cell responses and could be a promising strategy to cure chronically infected patients. 28 Symposium V – Chronic Viral Infections: HIV and Hepatitis ST 29 Impact of HLA‐B alleles on the immune evasion of hepatitis delta virus from CTL response and epitope discovery H. Karimzadeh1, B. Budeus2, A. Kosinska1, A. Heinold3, M. Buti4, M. Rizzetto5, D. Hoffmann2, U. Protzer1 M. Roggendorf1 1
Institute of Virology, Technical University of Munich/Helmholtz Center, Munich, Germany Department of Bioinformatics, University of Duisburg‐Essen, Essen, Germany 3
Institute for Transfusion Medicine, University of Duisburg‐Essen, University Hospital Essen, Essen, Germany 4
CIBERehd and Departments of Microbiology and Hepatology, Vall d'Hebron Hospital, University Autònoma de Barcelona (UAB), Barcelona, Spain 5
Department of Medical Sciences, University of Turin, Turin, Italy 2
Hepatitis delta virus (HDV) causes the most severe form of liver disease in hepatitis B carriers. Based on studies in animal models, DNA and/or Adeno‐vector vaccines could be protective against HDV by induction of virus‐
specific T cell response. However, little is known about the HDV‐specific CTL epitopes in human and whether their mutations may lead to viral evasion and adaptation. Therefore, we evaluated viral sequence variability in correlation with host HLA background as an approach to identify possible immune escape of HDV from T cells and consequently, using this approach for CTL epitope discovery on the large hepatitis delta antigen (L‐HDAg). The gene of L‐HDAg was amplified and sequenced from a cohort of 112 patients with chronic HDV infection from 6 European medical centers, and the HLA class I alleles were determined. Coincidence of certain residual substitutions and particular HLA class I alleles was tested. The binding affinities of the selected epitopes with and without substitutions to the corresponding HLA molecules were measured in vitro. We pinpointed immune selection pressure for 2 residues within a novel region restricted to HLA‐B*27 (aa 99‐108: RRDHRRRKAL). This HLA‐B*27‐restricted epitope was identified by its binding to HLA‐B*27 molecule in vitro and by induction of interferon secretion by CD8 T cells isolated from a patient with resolved HDV infection. A strong HLA footprint was also observed in the predicted novel HLA‐B*15‐restricted CD8 T cell epitope (aa 170‐179: SMQGVPESPF). A serine (S) to asparagine (N) substitution at position 1 of this region (S170N) was observed in all 8 (100%) HLA‐
B*15 positive patients whereas only 10% (11/112) of HLA‐B*15 negative group showed this substitution. This strongly indicated a possible immune escape of HDV at this position. Further analysis of correlations between substitutions throughout the whole 214 amino acids and 35 HLA class I alleles (locus A and B) confirmed a significant immune selection pressure at additional regions restricted to HLA‐B*13, ‐B*15, ‐B*37 and ‐B*41. Conclusion HLA‐B alleles seem to have a stronger selection pressure on HDV and, consequently, a greater impact on viral evolution than locus‐A of HLA does. Proper immune response to the CTL epitopes, especially to those located in conserved regions may support clearance of HDV infection e.g. the novel HLA‐B*27 restricted epitope. These results may be considered in HDV vaccine design studies with respect to HLA‐B alleles and possible selection pressure. 29 Emerging Infections and Pneumonia POSTER P 1 Acute Q‐fever infection after contact to fawn cadaver in Thuringia, a case report B. T. Schleenvoigt1, L. Sprague2, K. Boden3, U. Moog4, G. Schmoock2, W. Battefeld 5, H. Neubauer2, M. W. Pletz1 1
Universitätsklinikum Jena, Zentrum für Infektionsmedizin, Jena, Germany Friedrich‐Loeffler‐Institut, Jena, Germany 3
Universitätsklinikum Jena, Institut für klinische Chemie, Jena, Germany 4
Tiergesundheitsdienst , Schaf‐ und Ziegengesundheitsdienst, Jena, Germany 5
Universitätsklinikum Jena, Klinik für Innere Medizin III, Jena, Germany 2
A 48 years old man presented in July 2013 with fever and fatigue. Clinical examination revealed a reduced general condition, fever and sinus tachycardia. Basic laboratory testing showed thrombocytosis and marked elevation of C‐reactive‐protein. Chest X‐ray demonstrated the presence of an infiltrate in the right upper lobe, subsequently confirmed by computed tomography. There was no clinical improvement after initial antibiotic treatment with ceftriaxone. No pathogen was isolated from sputum or blood culture. Due to the non‐specific clinical picture further serologic investigation was performed (Brucella spp., Coxiella burnetii, Francisella tularensis, Borrelia spp., Leptospira spp., Listeria spp., Chlamydia spp., Mycoplasma und CMV). Based on positive serology results for Coxiella burnetii (IgG‐phase 2 ELISA: 41.1, IgG‐phase 1 ELISA: negative, IgA ELISA: negative, IgM ELISA: negative, IgG‐phase 2 IFT: 1:128, IgM‐phase 2: 1:64, IgG‐phase 1 IFT: negative) the diagnosis of acute Q‐fever with Q‐fever‐pneumonia was considered. Specific antibiotic treatment was initiated with ciprofloxacin 500 mg oral bid for 14 days. The patient responded well to treatment and fever subsided within 24 hours. Further serology tests performed after 2 and 8 months ruled out chronic coxiella infection. Retrospective anamnesis to clarify the origin of the infection revealed that 14 days before onset of acute disease the patient had buried two fawn cadavers. Analyses of soil samples taken from the burial site in September 2013 by real‐time PCR revealed the presence of C. burnetii‐DNA. The cadavers were not available for investigation due to decomposition and game damage. This is the first case report of an acute Q‐fever infection in which the source of infection can be linked to deer. 30 E m erg i ng nfections and Pneumonia
P2
3M R G N - plasmids of p andem i c S T 131 and S T 6 48 E. coli i nc rease bio lm production
possibly c ontributin to the p andem i c su ccess of these linea es i n numerous habitats
L. H. Wieler1, K. Sc aufler2, T. Semmler2,3, C . Ewers2, D. J . P ickard4 , G. Dougan4 , S. Günther2
1
Robert Koch-Institut, P rä sident, Berlin, Germany
Freie Universität Berlin, Institut für Mikrobiologie und Tierseuchen, Berlin, Germany
3
Robert Koch-Institut, NG 1, Berlin, Germany
4
Wellcome Trust, Sanger Institute, C ambridge, Great Britain
2
3MRGN-E .co li have become abundant all over the world, not only in a human clinical and community context
but also in animals and the environment. C lonal lineages of 3 MRGN-associated STs 131 and 6 4 8 are important
pandemic multi-resistant bacteria. This study aimed to investigate the influence of ESBL-plasmid-encoded nonresistance genes on chromosomally-encoded features of ST131 and ST6 4 8 E . co li. ESBL-plasmid carrying wildtype (WT) E . co li strains, their corresponding ESBL-plasmid-“ cured” variants (P C V) as well as complementary
ESBL-carrying transformants were comparatively analyzed in long-term colony, swimming motility and
Omnilog® P henotypic Microarray assays, whole-genome sequence and RNA sequence analysis. Differences
were detected in several phenotypic tests including an enhanced curli and/ or cellulose production and a
reduced swimming capacity of some ESBL-carrying strains compared to their P C V. Omnilog® results pointed
towards a similar metabolic behavior of the strains. RNA sequencing mostly confir ed the phenotypic results
on a genomic level, revealing the chromosomally-encoded cs g D -pathway as a key factor involved. P henotypic
differences, the reversibility in transformants and RNA sequencing results clearly indicate an influence of ESBLplasmids on chromosomally encoded features especially important for the subtle interactions between a
sessile and planktonic way of life in these 3MRGN E . co li, presumably contributing to their pandemic success.
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Emerging Infections and Pneumonia P 3 Potential target for broad spectrum antiviral drugs E. Acosta1, W. Fischl1,2, A. Ruggieri1, L. Kaderali3, H. Erfle4, S. Kallis1, R. Bartenschlager1,5 1
Universitätsklinikum Heidelberg, Zentrum für Infektiologie, Molekulare Virologie, Heidelberg, Germany Haplogen GmbH, Vienna, Austria 3
TU Dresden, Institut für medizinische Informatik und Biometrie, Dresden, Germany 4
Universität Heidelberg, ViroQuantCellNetworks RNAi Screening Facility, Heidelberg, Germany 5
Deutsches Zentrum für Infektiologie, Heidelberg, Germany 2
Emerging and re‐emerging infections are often caused by RNA viruses and are still a major public health concern as exemplified by recent outbreaks of MERS‐CoV in the Middle East or the current outbreak of Ebola virus in Western Africa.To establish a first‐line of defense against RNA virus groups that have the potential to cause epidemics and for which vaccines or selective therapies are not available, new broad‐spectrum antiviral drugs targeting viral structures or host cell factors that are conserved for a given virus group or family and that are essential for efficient virus replication are required. These drugs must have high selectivity, low toxicity and cover a broad class of viruses within a group or family. Dengue viruses (DENV) which belong to the family of Flaviviridae are mosquito‐borne emerging human pathogens whose infections produce a variety of clinical outcomes ranging from asymptomatic to the life‐
treating forms dengue hemorrhagic fever/dengue shock syndrome. The annual global incidence of dengue is estimated to reach 390 million infections per year. In spite of serious efforts, there are no approved therapies to prevent or inhibit DENV infections. By employing two sequential siRNA‐based high‐throughput screens employing DENV‐2 reporter virus we were able to identify peptidylarginine deiminase type IV (PADI4) as drug target being essential for the replication of all four DENV serotypes, West Nile virus and Hepatitis C virus. This strongly argues that PADI4‐inhibitors are suitable candidates for broad‐spectrum antiviral drugs. Currently, we are determining its clinical application by exploring the mode‐of‐action of the inhibitor and analyzing its efficacy against other virus groups employing the DZIF virus test pipeline (VTP). 32 Emerging Infections and Pneumonia P 4 Viral shedding and antibody response in 37 patients with MERS‐coronavirus infection V. M. Corman1, A. M. Albarrak2, A. S. Omrani2, M. M. Albarrak3, M. E. Farah4, M. Almasri5, D. Muth1, A. Sieberg1 B. Meyer1, A. M. Assiri5, T. Binger1, K. Steinhagen6, E. Lattwein6, J. Al‐tawfiq7, M. A. Müller1, Z. A. Memish8 C. Drosten1 1
University of Bonn Medical Centre, Institute of Virology, Bonn, Germany Prince Sultan Military Medical City, Division of Infectious Diseases, Riyadh, Kingdom of Saudi Arabia 3
Prince Sultan Military Medical City, Department of Critical Care, Riyadh, Kingdom of Saudi Arabia 4
Central Military Laboratory & blood bank, Microbiology Division, Riyadh, Kingdom of Saudi Arabia 5
Ministry of Health, Riyadh, Kingdom of Saudi Arabia 6
EUROIMMUN AG, Lübeck, Germany 7
Johns Hopkins Aramco Healthcare, Dhahran, Kingdom of Saudi Arabia 8
Alfaisal University, College of Medicine, Riyadh, Kingdom of Saudi Arabia 2
Background The MERS coronavirus causes isolated cases and outbreaks of severe respiratory disease. Essential features of the natural history of disease are poorly understood. Methods Here we studied 37 adult patients infected with MERS‐CoV for the development of viral loads in the lower and upper respiratory tract (LRT and URT), blood, stool, and urine. Antibodies and serum neutralizing activities were determined over the course of disease. Results 199 LRT samples collected throughout 3 weeks after diagnosis yielded virus RNA in 93% of tests. Average (maximum) viral loads were 5X10e6 (6X10e10) copies per mL. Viral loads (detection frequencies) in 84 URT samples were 1.9X10e4 cop/mL (47.6% positive). Viremia was not correlated with LRT viral load or the presence of neutralizing antibodies in serum. About half of all serum samples taken during the first week after diagnosis yielded viral RNA (33 % of all 108 sera tested). Only 14.6% of stool and 2.4% of urine samples yielded viral RNA. All seroconversions occurred during the first 2 weeks after diagnosis, corresponding to weeks 2 and 3 after onset of symptoms. IgM detection provided no advantage in sensitivity over IgG detection. All surviving patients but only slightly more than half of all fatal cases produced IgG and neutralizing antibodies. The levels of IgG and neutralizing antibodies were weakly and inversely correlated with LRT viral loads. Presence of antibodies did not lead to the elimination of virus from LRT. Conclusions The timing and intensity of respiratory viral shedding in MERS patients is very similar to that in SARS patients. Excretion in stool and urine is clearly less frequent and intense compared to SARS. Blood viral RNA seems to be not infectious but extrapulmonary places of virus replication seems possible. Neutralizing antibodies do not suffice to clear the infection. 33 Emerging Infections and Pneumonia P 5 Virological and serological monitoring of wild birds for zoonotic pathogens in Germany U. Ziegler1, D. Fischer2, K. Müller3, M. Rinder4, M. Eiden1, G. Dobler5, M. H. Groschup1 1
Friedrich‐Loeffler‐Institut, Institute of Novel and Emerging Infectious Diseases, Greifswald‐Insel Riems, Germany Justus Liebig University Giessen, Clinic for Birds, Reptiles, Amphibians and Fish, Giessen, Germany 3
Freie Universität Berlin, Department of Veterinary Medicine, Small Animal Clinic, Berlin, Germany 4
Ludwig Maximilians University Munich, Clinic for Birds, Reptiles, Amphibians and ornamental Fish, Centre for clinical veterinary medicine, Munich, Germany 5
Bundeswehr Institute of Microbiology, Department of Virology and Rickettsiology, Munich, Germany 2
West Nile virus (WNV) is a mosquito‐borne viral pathogen of global importance. In Europe clinical infections in humans and animals with WNV lineage 1 and/or 2 occur in Northern Italy, Southern France, Spain, in many Balkan countries (e.g. Hungary Croatia, Albania, Greece) as well as in Austria. An incursion of WNV into Germany is possible. The epidemic spread of a close WNV relative, the avian Usutu virus with low zoonotic potential to southwest Germany has provided the proof of principle for such an event in the last years. Because migratory birds play an important role in spreading of different zoonotic pathogens, we have therefore monitored migratory and resident birds by qPCR and by serology for flavivirus infections. In our two previous studies we investigated over 3,000 blood samples from wild birds. Antibodies to WNV were found in several migratory bird species, but WNV‐specific RNA has never been detected so far in any of these studies. These results are in accordance with our recently realised study from 2011 to 2013, where over 900 wild birds (representing 87 bird species belonging to 14 bird orders) have been bled and tested. Essentially our data show no evidence for indigenous WNV infections in the past, even though potential vector mosquitoes are present in Germany. However, our activities represent only a small part of the German wild bird population. For a better and extensive wild bird monitoring we will systematically set up a nation‐wide wild bird surveillance network for zoonotic arthropod‐borne virus infections (e.g. flaviviruses, influenza viruses, alphaviruses). Testing wild birds or sentinels in risk areas (mosquito‐rich wetlands, stream and marsh habitats, important stopover sites on the eastern and western flyways for migratory birds) as well as conducting monitoring programmes for mosquitoes and horses are useful approaches to monitor the human infection risk for pathogens in these areas. Due to trade and climate change effects and the establishment of exotic mosquitoes (Aedes japonicus, Aedes albopictus), the risk of introduction of new zoonotic pathogens into Germany is increasing steadily. 34 Emerging Infections and Pneumonia P 6 DiAL‐FISH for the rapid detection and identification of bacterial agents K. Aistleitner1, K. Stoecker1, T. Sieper1, I. Stürz1, R. Wölfel1 1
Institut für Mikrobiologie der Bundeswehr, Munich, Germany Fluorescence in situ hybridization (FISH) is a powerful method for the cultivation‐independent in situ detection and identification of microorganisms. Since its establishment over two decades ago it has become an essential tool in microbial ecology. As it is an easy, robust, cheap and rapid method, FISH is also occasionally used in clinical settings. However, until recently only three bacterial species could be identified simultaneously in one hybrization step, making the identification of larger sets of bacterial species laborious and time‐consuming, thereby impairing the use of FISH in many diagnostic approaches. Here we report on the development of an rRNA‐targeted FISH based diagnosticalgorithm (DiAL‐FISH) allowing for the cultivation‐independent rapid detection, identification and quantification of up to thirteen bacterial pathogens in clinical samples. Novel group‐specific probes targeting members of the Rickettsiaceae, the Bacillus cereus group and Leptospiraceae as well as species‐specific probes targeting Vibrio cholerae, Yersinia pestis, Escherichia coli and Coxiella burnetii were designed using the arb‐software package and evaluated using formamide‐series and the daime software. By combing these probes with previously published probes for Brucella spp, Burkholderia mallei, Burkholderia pseudomallei, Neisseria meningitidis, and Francisella tularensis the diagnostic assay now targets thirteen bacterial pathogens. Using multicolored double‐labeled oligonucleotide probes, these species can now be identified by only two hybridizations in less than four hours. The algorithm was successfully applied for the identification of infectious agents in various sample materials, including paraffin sections of lymph nodes, powder samples and skin surfaces. To overcome the need for a fume hood and allow for broader application of this method, we currently aim to substitute for toxic formamide, which is used in conventional FISH to adjust the stringency of the probe, by non‐toxic urea. Interestingly, usage of urea so far not only results in the specific detection of bacteria, but also in brighter fluorescence signals for some probes. In summary, DiAL‐FISH allows the fast and direct visualization of rarely occurring, but important bacterial pathogens in a straightforward and robust manner. In addition, it provides a different and independent laboratory method that supplements PCR‐based detection methods for these bacteria. 35 Emerging Infections and Pneumonia P 7 Identification of a novel pathogenic Enterobacter species: determining virulence factors using next generation sequencing. N. B. Pati1, S. P. Doijad1, L. Falgenhauer1, J. Overmann², Y. Yao1, T. Hain1, C. Imirzalioglu1, T. Chakraborty1 1
Institute of Medical Microbiology, German Centre of Infection Research, Site Giessen‐Marburg‐Langen, Justus‐Liebig‐
University Giessen, Giessen, Germany ² Leibniz Institute DSMZ‐German Collection of Microorganisms and Cell Cultures, and German Centre of Infection Research (DZIF), Partner Site Hannover‐Braunschweig, Braunschweig, Germany Enterobacter species are nosocomial pathogens and a leading cause of neonatal sepsis worldwide. We previously described an outbreak caused by a hitherto undescribed multidrug resistant Enterobacter species in a neonatal unit of a tertiary care hospital in east Africa. Using vertebrate and invertebrate models of infection we found Enterobacter species 247 (E247) to be highly pathogenic. Whole genome sequencing and biochemical characterization confirmed E247 to represent a novel species that harbors T6SS, multi copy genes regulating O‐antigen synthesis and transport, gene clusters responsible for capsule production and a large number of genes for iron uptake and metabolism. E247, unlike other Enterobacter spp., grew in high concentrations of human serum indicating septicemic potential. We identified genes mediating serum resistance by whole genome‐based transcriptomics. We found that 7% of the whole genome was mobilized to enable growth in this environment. In particular, differential expression of genes contributing to iron uptake, transport and energy metabolism was observed which was further confirmed by qRT‐PCR. We also found that the O‐antigen is involved in serum survival and cytokine induction. To overcome the therapeutic limitations of such an MDR strain we targeted iron uptake mechanisms using lasso peptide (MccJ25), a microcin with affinity to FhuA that is a potent inhibitor of iron uptake in E. coli. Growth of E247 was inhibited by MccJ25 suggesting an alternative to the current therapeutic strategies. In conclusion, our work has demonstrated the successful application of next generation sequencing for rapid virulence factor assessment of novel bacterial pathogens. Using the whole genome sequencing approach we identified E247 to be a novel virulent Enterobacter species carrying multiple virulence genes. Essentially, the transcriptomic approach has provided a comprehensive idea about the necessary gene sets that are involved in E247 adaptation to human serum and causing disease. Future work will reveal more about the molecular mechanisms behind the pathogenesis of this novel Enterobacter pathogen. 36 Emerging Infections and Pneumonia P 8 Changes in risk perceptions related to the 2014 Ebola virus disease epidemic: results of two consecutive surveys in Lower Saxony N. Rübsamen1,2, E. Garsevanidze2, S. Castell2, J. Horn1,2, G. Krause3,2, J. Ott3,2, H. Raupach‐Rosin2, A. Karch3,1,2 R. T. Mikolajczyk3,2 1
PhD Programme “Epidemiology", Braunschweig‐Hannover, Germany Helmholtz Centre for Infection Research, Department of Epidemiology, Braunschweig, Germany 3
German Centre for Infection Research, Hannover‐Braunschweig site, Braunschweig, Germany 2
Background Individual risk perceptions and subsequent changes in behaviour can play an important role for the dynamics of epidemics and need to be taken into account in risk communication and for the implementation of public health measures. Only limited information is available about individual risk perceptions and behavioural changes during pandemic threats. We assessed risk perceptions and behaviour of the German population at two time points during the 2014 Ebola virus disease (EVD) epidemic in West Africa. Methods In November 2014, we surveyed the participants of a longitudinal online panel addressing hygiene and preventive behaviour regarding infections (HaBIDS) in four districts of the federal state of Lower Saxony regarding their risk perceptions related to the EVD epidemic in West Africa. Of 1,376 adults aged 15 to 69 years, randomly sampled from the residents’ registration offices and enrolled in the panel, 974 (70.8%) agreed to participate. The questionnaire covered risk perceptions, knowledge about transmission routes of EVD, media use, personal reactions to the outbreak, attitudes towards measures to prevent the spread of EVD to Europe, and attitudes towards a potential vaccination against EVD. In August 2015, we sent a follow‐up questionnaire to all 2014 participants; 258 of the 974 (26.5%) agreed on a repeated participation. Results In 2014, 26.1% of the participants expressed personal worries about EVD; this proportion dropped considerably to 2.0% in the second survey. While the level of knowledge regarding EVD decreased in the majority of participants (52.1%) between the two time points, 26.7% had improved their knowledge in 2015 compared to 2014. Increase in knowledge was positively associated with being worried about EVD in 2014 (p = 0.03). In the hypothetical scenario of an EVD patient coming to Germany for treatment in a nearby hospital, 83.0% of all participants reported in 2014 that they would adopt additional preventive behaviours. This included avoidance of public transportation (13.4%), improvement of individual hygiene behaviour (62.0%), and the decision not to visit relatives admitted to the same hospital (17.8%). These modifications of behaviour were reported virtually unchanged in the 2015 survey. Conclusion Although personal worries about EVD decreased after the peak of the epidemic, the scenario of an EVD patient being treated nearby is still associated with considerable behavioural changes among participants. Change in knowledge between the two time points is associated with the level of worries expressed in 2014, indicating that people who were initially worried were more likely to acquire information about EVD than people who were less worried. Our findings underline the importance of providing clear and easily accesible information about transmission routes and the risk of acquiring pandemic diseases in countries currently not affected in order to avoid unrealistic risk perceptions. 37 Emerging Infections and Pneumonia P 9 High prevalence of malaria and gramnegative bloodstream infection in febrile outpatients attending a rural hospital in Southwestern Tanzania N. Heinrich1,2, B. Flach2,3,4, C. Mangu5, H. Msila5, L. Maboko5, H. Schimanowski ‐ Thomsen6, C. Mwansongela6 B. Hoehne1,2, G. Dobler2,3,4, M. Hoelscher1,2 1
Klinikum der Universität München, Abteilung für Infektionskrankheiten und Tropenmedizin, Munich, Germany German Center for Infection Research (DZIF), Munich Partner site, Munich, Germany 3
Bundeswehr Institute of Microbiology, Munich, Germany 4
German Partnership Program for Excellence in Biological and Health Security, Munich, Germany 5
NIMR ‐ Mbeya Medical Research Centre, Mbeya, Tanzania 6
Matema Lutheran Hospital, Matema, Tanzania 2
Background Febrile illness is one of the major contributions of hospital admissions and death in Sub‐Saharan Africa. We present interim results from an ongoing study on febrile disease etiology, socio‐economic and environmental covariates in a rural location in South‐Western Tanzania, that is jointly conducted by the German Partnership Program for Excellence in Biological and Health Security, by DZIF and by NIMR ‐ Mbeya Medical Research Centre. Methods Individuals with temperature >37.5°C, and aged > 1 year, are being enrolled into the study. All patients received clinical assessment, rapid HIV and Malaria testing, urinalysis, blood culture. Positive blood cultures were differentiated by bacterial morphology and biochemical reactions. Results 242 patients have been enrolled to this point. 67.5% of all enrolled patients were <18 years, and 55.2% were female. Body temperature ranged from 37.5°C to 41.1°C (Median 38.4°C) and duration of fever was reported from 1‐30 days (Median 3 days). At the initial assessment, 101 cases received more than one diagnosis by the attending clinician. Main diagnoses included rapid test confirmed malaria (n=145), fever of unknown origin (n=33), tonsillitis/pharyngitis (n=29), lower respiratory tract infection (n=28); upper respiratory tract infection (n=21), diarrhea (n=16), urinary tract infection (n=14), abdominal infection (n=13), otitis media (n=9). 20% of all patients tested positive for HIV. 63.5% of patients tested positive for Malaria, of which 47% were P. falciparum, 51% other malaria forms, and 2% mixed infections according to the rapid test. 13.2% of all enrolled patients showed a positive blood culture. Preliminary identification of gram negative bacteria via colony morphology and biochemical reactions suggests a high number of Salmonella enterica (37% of positive blood cultures). Serotyping to differentiate typhoid from non‐typhoid organisms is pending. 24% of all acutely febrile patients were negative in all diagnostic tests performed. Those samples will need to be investigated further. Conclusion To date, the cohort shows a surprisingly high positivity in malaria diagnostic tests. Of concern is a high blood culture positivity in this ambulatory patient collective. Further analysis is ongoing. 38 Emerging Infections and Pneumonia P 10 Introducing a new inter‐disciplinary network on Taenia solium cysticercosis and taeniosis research in sub‐Saharan Africa: CYSTINET ‐ Africa V. Schmidt1, H. Ngowi2, B. Ngowi3, S. G. Mfinanga3, C. S. Sikasunge4, I. K. Phiri5, K. E. Mwape5 E. Normahoomed6, B. Brügge7, C. Prazeres da Costa8, A. S. Winkler1 1
Technical University Munich (TUM), Department of Neurology, Munich, Germany Sokoine University of Agriculture, Department of Veterinary Medicine and Public Health, Morogoro, Tanzania 3
National Institute for Medical Research (NIMR), Medical Research Centre (MMRC), Dar es Salaam, Tanzania 4
School of Veterinary Medicine, The University of Zambia (UNZA), Department of Paraclinical Studies, Lusaka, Zambia 5
School of Veterinary Medicine, The University of Zambia (UNZA), Department of Clinical Studies, Lusaka, Zambia 6
Medical Faculty, Eduardo Mondlane University (EMU) & Mozambique Institute for Health Education and Research (MIHER), Department of Parasitology, Maputo, Mozambique 7
Technical University Munich (TUM), Department of Informatics, Munich, Germany 8
Technical University Munich (TUM), Institute for Medical Microbiology, Immunology and Hygiene (MIH), Munich, Germany 2
Project description Taenia solium cysticercosis/taeniosis (TSCT), an emerging but neglected zoonosis, represents a potentially eradicable disease complex in many countries of sub‐Saharan Africa with huge impact on human and animal health. To fill gaps on the way to control/elimination of TSCT an interdisciplinary research platform will be developed integrating basic and epidemiological research, capacity building and international networking. Four African partners ‐ one in Mozambique (M), two in Tanzania (T) and one in Zambia (Z) ‐ and two German partners (both TU Munich) are involved and linked with an international advisory board including the WHO, CDC Atlanta, CYSTINET‐Europe. A strong focus within CYSTINET‐Africa is to define immunological determinants of host‐parasite interaction which will foster the development of basic science within that area. Furthermore, international conferences and the development of an IT platform that comprises a virtual one‐health center with a research school as well as tools for electronic case management and networking are other key elements. CYSTINET ‐ Africa will officially start in 2016 and has received funding from the German Federal Ministry of Education and Research (BMBF) for activities planned until 2021. Specific research activities ‐ Assessment of prevalence and co‐infections of TSCT/neurocysticercosis (NCC) in large‐scale community based studies in T,M,Z ‐ Evaluation of the pathomechanisms involved in the development of symptomatic NCC in immunocompetent and immunocompromised individuals as well as pathomechanisms involved in different treatment responses in people with symptomatic NCC on standard treatment in longitudinal studies in T and M ‐ Establishment of an experimental pig model for T. solium cysticercosis for human and veterinary research needs and investigation of pig breed susceptibility to T. solium in Z ‐ Development, evaluation and implementation of a low‐cost health education intervention package for the prevention and control of TSCT in T Project outlook CYSTINET‐Africa will create impact by linking human, animal and community health workers (based on the “one‐health” principle) with respect to zoonotic diseases at various levels in sub‐Saharan Africa. A rapid expansion of this network and its activities is envisaged and promising new collaborations are already on the way with teams in Uganda, Zimbabwe and Madagascar. 39 Emerging Infections and Pneumonia P 11 Tick‐borne encephalitis in Germany ‐ Actual Situation and Trends G. Dobler1,2 1
2
Institut für Mikrobiologie der Bundeswehr, Virologie und Rickettsiologie, Munich, Germany DZIF German Center of Infection Research, Partner Site Munich, Munich, Germany Tick‐borne encephalitis (TBE) is the most important arbovirus disease in Europe. It is caused by the tick‐borne encephalitis virus (TBEV). In Central Europe, including Germany, exclusively the European subtype has been detected in ticks so far. In Germany between 200 and 400 human TBE cases are registered every year. The main endemic areas of TBE are located in southern Germany in the federal states of Bayern, Baden‐Württemberg and in parts of Thuringia, Hesse, Saxonia and Rhineland‐Palatine. However, during the last years sporadic reports of autochthonous human cases were also submitted from other federal states and the TBEV could be detected in ticks in Mecklenburg‐Prepommerania and Saxonia‐Anhalt. The registered and confirmed (SurvStat, RKI, Berlin) human cases of TBE from 2001 to 2014 were analyzed according to prevalence and trend of occurrence to study the recent spread of TBE and possible tendencies of TBE spread in Germany. Furthermore, the human TBE cases of highly endemic districts in Bavaria were analyzed in detail to study trends in prevalence of human disease in relation to human vaccination against TBE. The analysis of human TBE cases in Germany shows a slightly increasing trend. On the level of German Federal States, there are clear trends of increasing levels in the State of Bavaria and of Saxonia, while in the other Federal States namely in Baden‐Württemberg, the analysis shows a slight increase from 2001 to 2008, following an decrease to the level of 2001. On the district level in Bavaria there is a general increase of trend in the districts of Amberg (the district with the highest incidence of TBE in Bavaria and Germany. But there are also increasing levels of TBE cases in parts of Middle Franconia while in Lower Franconia, a classical endemic area the seems to be a trend of stabilization or even decrease in some of the former highly endemic districts. Recently a clear increase of TBE in some parts of the Federal State of Saxonia was detected, resulting in the classification of the district of Vogtland as endemic area in 2014. TBE is still a big medical problem in parts of Southern Germany. There seems to be a clear trend of increasing human TBE cases in Bavaria while in Baden‐Württemberg there was a recent consolidation on high level. This is even more interesting as the vaccination rates in Bavaria are higher than in Baden‐Württemberg. So far it is unclear what reasons cause this increase of human TBE cases in Bavaria. There seems to be an increase of establishment of new natural foci in some of the studied districts. But also social effects, like increasing activities in nature and the increasing invasion into established natural foci may contribute to the increasing trend of TBE in Bavaria. As the natural foci cannot be eradicated only a universal TBE vaccine in Federal States with known endemicity may finally lower the TBE incidence. 40 Emerging Infections and Pneumonia P 12 Phylo‐geography of Tick‐borne Encephalitis Virus in Austria and the Neighboring Countries M. Bestehorn1,2, L. Chitimia‐Dobler1,2, F. X. Heinz3, K. Stiasny3, G. Dobler1,2 1
Institut für Mikrobiologie der Bundeswehr, Virologie und Rickettsiologie, Munich, Germany DZIF Deutsches Zentrum für Infektionsforschung, Partner Site Munich, Munich, Germany 3
Medizinische Universität Wien, Institut für Virologie, Vienna, Austria 2
Tick‐borne encephalitis virus (TBEV) is the most important tick‐borne virus in Europe and Asia. Recent studies show that the origin of TBEV lies in the Siberian region, from where it spread into Central Europe more than 3000 years ago. New data show that there is also an actual movement of TBE endemic areas with human TBE cases disappeared from former highly endemic areas and emerged in new areas as shown recently in Austria. There, human cases disappeared in Eastern Austria and newly emerged in the federal states of Tyrolia and Vorarlberg (Heinz et al. 2015). To understand the spreading of TBEV in Austria and the neighboring countries, E gene sequences of TBEV strains from recently emerged new endemic areas in Austria, Slovakia and SE Germany were analyzed and compared to E gene sequences of former isolates in data bases. A phylo‐geographic analysis was conducted forming a relation of genetics and geographic places of occurrence. The so far available data show that the Austrian isolates from the 1970s and 1980s cluster in phylogenetic arbitrary genotypes with viruses from Germany, the Czech Republic and the Slovak Republic. The next closest genetic relative of a new TBE virus strain isolated from Tyrolia in 2015 and strains from actual TBEV strains from Bavaria however is a TBEV strain from Finland (strain Joutseno). This observation shows that the recently emerged TBE endemic areas in Austria and Germany are not formed by continuous spread of TBEV strains from existing natural foci along natural landscape aisles (e.g. river valleys of Ziller and Inn), but seem to be recently introduced from other regions (Northern Europe). The mode of introduction is unclear, however recent data from Germany suggest that migratory birds may play an important role in the recent spread of TBEV in Europe and the emergence in new regions. 41 Emerging Infections and Pneumonia P 13 Identification of Rickettsiae from ticks collected in cattle in South‐Western Tanzania B. Flach1,2,3, M. Knüpfer4,2,3, K. Baumann1,2,3, L. Chitimia‐Dobler4,2,3, N. E. Ntinginya5, L. Maboko5, N. Heinrich3,6 M. Hölscher3,6, G. Dobler1,2,3 1
Bundeswehr Institute of Microbiology, Department for Virology and Rickettsiology, Munich, Germany German Partnership Program for Excellence in Biological and Health Security, Munich, Germany 3
German Center for Infection Research, Munich Partner Site, Munich, Germany 4
Bundeswehr Institute of Microbiology, Munich, Germany 5
NIMR‐Mbeya Medical Research Centre, Mbeya, Tanzania 6
Division for Infectious Diseases and Tropical Medicine, Medical Center of the University of Munich, Munich, Germany 2
Background Rickettsiae are fastidious intracellular bacteria, transmitted by arthropod vectors, causing mild to severe diseases in humans and animals. Rickettsiae from the spotted fever group (SFGR), as well as their vectors have been described in some countries in Africa, but not much is known about the distribution in Tanzania. We have recently identified high seroprevalences against SFGR in 9 different communities in the Mbeya region. The seroprevalences were 77.6% and 81.4% in the regions of Utengule and Igurusi, respectively. In this study, the tick species composition on cattle and the prevalence of rickettsiae in ticks were tested. Methods: Ticks were collected from cattle markets, identified using taxonomic keys and 16s PCR. Tick DNA was extracted and screened for rickettsiae using a qPCR assay that amplifies a 100 bp fragment of the conserved citrate synthase gene. All Rickettsia‐positive tick DNA samples were amplified and sequenced with primer sets that target rickettsial genes and the 23S rRNA. Results 584 ticks were collected and members of four tick genera were identified, with Rhipicephalus (Rh.) being the most abundant (65.4%), followed by Amblyomma (A., 28.4%), Hyalomma (H., 6.0%) and Haemaphysalis (0.2%). We classified 21 species in all four genera. Species previously not described in Tanzania were found for the first time, e.g. Rh. geigyi and Rh. annulatus, H. dromedarii and H. marginatum, as well as A. pomposum. 51% of ticks tested positive for rickettsiae (R.) infection. Of infected ticks, 47% belonged to the Amblyomma generus, followed by Rhipicephalus (43,1%). Positivity in both species were 84,3% and 34%, respectively. After sequencing, R. africae was found to be the dominant rickettsiae species, mostly carried by A. variegatum (64,7%) and Rh. microplus (21,3%). R. africae was also found in R. geigyi and H. marginatum (1.6%). Unexpectedly, we found one Rh. evertsi infected with R. massiliae, a pathogen previously only described in Northern and West Africa. Conclusion To our knowledge, this is the first study describing distribution of rickettsiae and its vectors in South‐Western Tanzania. High human seroprevalence and the high number of infective ticks show the potential medical importance of rickettsiosis in Tanzania and the necessity for a raised awareness. The detection of previously unknown tick and Rickettsiae species in Tanzania also addresses the need for further investigations of distribution and prevalence in East Africa. 42 Emerging Infections and Pneumonia P 14 Evidence for an ancestral association of human coronavirus 229E with bats V. M. Corman1, C. Drosten1, J. F. Drexler1 1
University of Bonn Medical Centre, Institute of Virology, Bonn, Germany The ancestral origins of major human coronaviruses (HCoV) likely involve bat hosts. Here, we provide conclusive genetic evidence for an evolutionary origin of the common cold virus HCoV‐229E in hipposiderid bats by analyzing a large sample of African bats and characterizing several bat viruses on a full genome level. Our evolutionary analyses show that animal and human viruses are genetically closely related, can exchange genetic material and form a single viral species. We show that the putative host switches leading to the formation of HCoV‐229E were accompanied by major genomic changes including deletions in the viral spike glycoprotein gene and loss of an open reading frame. We re‐analyze a previously described genetically related alpaca virus and speculate on the role of camelids as potential intermediate hosts between bat and human viruses. The evolutionary history of HCoV‐229E likely shares important characteristics with that of the recently emerged highly pathogenic MERS‐Coronavirus. 43 Emerging Infections and Pneumonia P 15 Strong immunity and protection of vaccinated animals induced by a measles virus‐based MERS‐vaccine A. Fiedler1,2, S. Prüfer1, A. Kupke3,4, V. A. Scheuplein 1, S. Hutzler1, D. Kreuz1, T. Beissert5, S. Bauer 1 S. Hubich‐Rau6, C. Tondera1, H. Shams Eldin3,4, J. Schmidt3,4, J. Vergara‐Alert3,4, Y. Süzer1, J. Seifried1 K.‐ M. Hanschmann1, U. Kalinke7,8, S. Herold9, U. Sahin5,6, K. Cichutek1,2, Z. Waibler1,2, M. Eickmann3,4 S. Becker3,4, M. D. Mühlebach1,2 1
Paul‐Ehrlich‐Institut, Langen, Germany German Center for Infection Research, Langen, Germany 3
Philipps Universität, Marburg, Germany 4
German Center for Infection Research, Marburg, Germany 5
TRON GmbH, Mainz, Germany 6
Universitätsmedizin, Mainz, Germany 7
Hannover Medical School and the Helmholtz Centre, Hannover, Germany 8
German Center for Infection Research, Hannover, Germany 9
Universities Giessen & Marburg Lung Center (UGMLC), Giessen, Germany 2
Viral diseases like the Spanish Flu have unexpectedly been emerging and are frequently able to cause severe pandemics with significant fatality rates. To prevent the spreading of emerging viruses and the occurrence of pandemics, fast available vaccines are required. The development of platform‐based, safe and easily producible vaccines may be an alternative to classic vaccine development. Replicating recombinant measles virus (MV) constitutes a promising tool to induce immunity also against other pathogens. The integration of antigen‐
coding genes into MV and consequentially the co‐expression of MV and foreign antigens can be achieved reasonably easy and quick. The Middle East Respiratory Syndrome coronavirus (MERS‐CoV) emerged firstly in spring 2012 and still causes considerable morbidity and mortality in patients. The ongoing occurrence of MERS‐
CoV outbreaks mandates the development of a vaccine also to counteract pandemic developments. Thus, we aimed to generate and characterize MVs expressing the spike glycoprotein (S) of MERS‐CoV in vitro and in vivo. Therefore, full length genes of MERS‐S and a truncated version encoding soluble S variant (MERS‐
solS) were cloned into two different additional transcription units (ATUs) of vaccine strain MVvac2. These MVs were rescued, amplified, and characterized in passage 3 and 10. The characterization revealed that the replication of all recombinant viruses in Vero cells was comparable to the control virus MVvac2‐GFP. Additionally, the genomic stability and expression of the inserted antigens was confirmed via sequencing of viral cDNA and immunoblot analysis. In vivo, immunization of IFNAR‐/‐‐CD46Ge mice with twice 1x105 TCID50 MVvac2‐MERS‐S(H) or MVvac2‐MERS‐solS(H) induced robust levels of antibodies directed against MV and MERS‐
CoV with neutralizing capacity. Additionally, induction of specific T cells was demonstrated by antigen‐specific T cell proliferation, cytotoxicity, and IFN‐g secretion. These immune responses protected vaccinated animals from challenge infection with MERS‐CoV shown by a significant reduction of viral loads as well as reduced MERS‐induced inflammation in the lungs of challenged mice. Thus, recombinant MVs expressing MERS‐CoV S antigen variants are a stable vaccine that induce both significant humoral and cellular immune responses and protect animals from infection with MERS‐CoV. These data demonstrate the versatility of the MV vaccine platform to combat emerging infections. 44 Emerging Infections and Pneumonia P 16 Rickettsiae in Ticks from Romanian Carnivores L. Chitimia‐Dobler1,2,3, G. Dobler1,2 1
Institut für Mikrobiologie der Bundeswehr, Virologie und Rickettsiologie, Munich, Germany DZIF Deutsches Zentrum für Infektionsforschung, Partner Site Munich, Munich, Germany 3
Institute of Diagnosis and Animal Health, Dept. of Parasitology, Bucarest, Romania 2
Rickettsiae are gram‐negative obligate intracellular bacteria and are exclusively vector‐borne. Romania due to the central Carpathian Mountains can be divided three climatic zones, the Atlantic‐influenced climate in the western part, a continental climate in the Northeastern part and a maritime (Mediterranean) climate in the Southeastern part of the country. The climatic conditions may have an important influence on the occurrence of ticks and of tick‐borne pathogens. Therefore the aim of the current study is to determine the tick species and their pathogens on dogs and foxes in different regions of Romania. Ticks were collected from foxes (national rabies program) and from dogs (veterinary cabinets) in nine districts in southern and western Romania. Ticks were tested as single specimen or in pools (up to 20 larvae, up to 6 nymphs or adult ticks using a panRickettsia real‐time PCR. PCR‐positive supernatants were further analyzed to identify the Rickettsia (Ri.) species by sequencing amplicons using a 23S RNA conventional PCR and sequencing of ompA, ompB partial genes. A total of 919 ticks were collected and tested. 315 ticks were collected from foxes and 604 ticks were collected from dogs. Three tick species were detected on foxes: Ixodes (Ix.) crenulatus, Dermacentor (De.) reticulatus and Ix. ricinus. All Ix. crenulatus and all De. reticulatus ticks from foxes gave negative results. 15/26 pools of Ix. ricinus were positive in the panRickettsia‐RT‐PCR for Ri. helvetica and Ri. monacensis. In dogs Rhipicephalus sanguineus, De. reticulatus and Ix. ricinus were detected positive for rickettsiae. Rickettsia‐positive ticks were only found in the northwestern part of Romania, a region which is in the influence of the Atlantic climate. The minimal infection rates for rickettsiae ranged from 7.4 to 12.1%. The identification of the rickettsiae in Rhipicephalus sanguineus resulted in the first ever detection of Ri. massiliae. Also Ri. helvetica and Ri. monacensis and Ri. raoultii were detected. Our results of ticks from dogs show an unequal distribution of rickettsiae in ticks in Romania with much higher prevalence rates in the northwestern part than in the southeastern part. It is however unclear what factors might be responsible for this distribution of rickettsiae in ticks and if the climatic conditions might be one of the important factors. Furthermore, the distribution of other tick‐borne pathogens in different parts of Romania should be further investigated. 45 Emerging Infections and Pneumonia P 17 Ixodes gibbosus a New Invasing Mediaterranean Tick Species in South‐eastern Germany L. Chitimia‐Dobler1,2, M. Bestehorn1,2, G. Dobler1,2 1
2
Institut für Mikrobiologie der Bundeswehr, Virologie und Rickettsiologie, Munich, Germany DZIF Deutsches Zentrum für Infektionsforschung, Partner Site Munich, Munich, Germany Ixodes (Ix.) gibbosus (Nuttall, 1916) is a tick species from the Ixodes genus and closely related to Ix. ricinus and Ix. persulcatus. The known geographic distribution of Ix. gibbosus are the warm and dry environments of some Mediterranean countries like Greece, Turkey and Spain. The warm and dry places are not well suitable for Ix. ricinus and, on the other hand, the shorter life cycle of Ix. gibbosus will substitute and replace the first species. So far, no data are available about the potential role of the species Ix. gibbosus as a vector of pathogens. However, it was considered to be responsible for some cases of tick‐paralysis in animals in the Mediteranean. During the sampling of ticks in a putative tick‐borne encephalitis virus natural focus South‐eastern Germany in June 2015, some morphologically distinct ticks were detected. Further morphological analysis of the ticks resulted in the identification of Ix. gibbosus. Molecular analysis using the sequence of mitochondrial Cox1 gene showed relation to but was clearly distinct from Ix. ricinus. Further sampling in the surroundings of the primary location could confirm the presence of this tick species and show that its distribution area seems to be larger and even a couple of this tick species was detected. Our data show that Ix. gibbosus, a tick species known to occur so far only in the Mediterranean region, developed a stable population in Eastern Bavaria. As Ix. gibbosus was found so far in a known TBEV focus, its role in the TBEV cycle has to be discussed. Also the potential of transmission of other pathogens like borreliae or rickettsiae has not been clarified at all for this tick species. The way and the time of introduction of Ix. gibbosus into Germany are unclear so far. It is also not known whether any, so far non‐detected pathogens were introduced together with the tick. The future prospects are to detect Ix. gibbosus in other places in Germany and its neighboring countries and to investigate its potential role as a vector of autochthonous and newly imported pathogens. 46 Emerging Infections and Pneumonia P 18 Extending distribution of Dermacentor reticulatus in Romania L. Chitimia‐Dobler1 1
Institute of Diagnosis and Animal Health, Dept. of Parasitology, Bucarest, Romania Dermacentor (D.) reticulatus (Fabricius, 1794), also known as the marsh tick or ornate dog tick is next to Ixodes ricinus the second most significant vector of tick‐borne diseases in Europe. It is the vector of different pathogens among them protozoa, rickettsiae and viruses. However, so far only very limited information on the distribution of D. reticulatus in Romania is available. Few surveys on the geographical distribution of ticks in Romania were conducted in the 1960s and then again in the 2000s. None of the 2000s’ studies have brought new data regarding the presence of D. reticulatus after Feider’s early study from 1965. A field survey monitoring the geographical distribution and periodic appearance of D. reticulatus in Romania was carried out in 18 counties from 2012 to 2015 in order to gain current data on the distribution and activity of D. reticulatus in Romania compared to former studies. Ticks were collected from dogs, foxes, sheep, cattle and wild boar which were available for other investigations. Part of the ticks was also flagged in 3 counties, in ecological areas suitable for this species. Altogether, 897 adult D. reticulatus ticks were collected, 442 of which were collected by flagging (231 females and 211 males) and 455 from animals: 380 from dogs (239 females and 141 males), 10 from cattle (6 females and 4 males), 4 from sheep (a female), 60 from wild animals: 52 from foxes, (19 females and 33 males), 8 males from a wild boar, and one female feeding on a human. In the current study, D. reticulatus was detected in 18 counties of which in 16 for the first time at all. This is in contrast to Feider's classical work from 1965 where he described this tick species only in three counties. The present survey provides evidence that this tick species has largely extended its geographical range since 1965 or that it was not correctly identified in the past. Furthermore, new tick‐host association of D. reticulatus is reported by the current study, showing new hosts and new data on the periodic activity of this tick species. 47 Emerging Infections and Pneumonia P 19 Comprehensive diagnosis and treatment of Alveolar Echinococcosis (AE) in Ulm/Germany: A single‐center experience of 112 patients (2012 to 2015) B. Grüner1, A. Hillenbrand2, J. Schmidberger3, W. Kratzer3, T. Gräter4, T. Barth5 1
Universitätsklinkum Ulm, Klinik für Innere Medizin III, Ulm, Germany Universitätsklinikum Ulm, KLinik für Allgemein‐ und Viszeralchirurgie, Ulm, Germany 3
Universitätsklinikum Ulm, Innere Medizin I, Ulm, Germany 4
Universitätsklinkum Ulm, Klinik für Radiologie, Ulm, Germany 5
Universitätsklinikum Ulm, Institut für Pathologie, Ulm, Germany 2
Background Human alveolar echinococcosis (AE) caused by Echinococcus mulitlocularis is rare, but increasing in Europe. 118 human AE‐cases were reported to the RKI from January 2012 to July 2015. Diagnosis and treatment are often a challenge. We report our interdisciplinary approach to patients referred with AE‐diagnosis to the specialized clinic at the University Hospital Ulm. WHO‐ PNM classification was the basic for treatment decisions according guidelines of the expert consensus of the WHO Informal Working Group on Echinococcosis. Patients 117 AE‐ patients were treated at Ulm University 01/2012‐07/2015. According to the WHO case definition AE was “confirmed” in 71 and “probable” in 41 patients. 5 “possible cases” were excluded. 65 were female and 47 male, median age at first diagnosis was 51 years (range 11 to 82). 2 patients presented with stage I, 35 with stage II and 15 with stage IIIA, meaning a good chance for complete resection of the AE‐lesion. Over 50% presented in advanced stages: 33 patients IIIB (extrahepatic involvement or P4 lesion (extension along vessels and biliary tree); 27 presented with stage IV, (P4 lesion + extrahepatic involvement or presence of distant metastasis, meaning cure is unlikely for those patients. Treatment In patients with “probable” or “confirmed” AE benzimidazole (BMZ) treatment was generally advised (n= 110) but for 2 we suggested watch and wait. In advanced AE the albendazole (ABZ) treatment was advised to be long‐term, whereas after complete resection BMZ for 2 years were given. 45 patients underwent surgery, i.e., 34 in curative intent (safety margin > 1 cm; additional 7 patients had a complete removal of their lesions with a safety margin < 1 cm). 4 patients with palliative resection are under BMZ long‐term treatmenz now. Reasons were haemorrhage, pain and also lack of knowledge regarding the therapy recommendations. 14 patients had ABZ intolerance, and 8 did not tolerate BMZ at all. All 112 patients are alive. 35 of 45 patients after surgery are regarded as cured. Conclusion PNM staging is condition for a structured therapeutic approach to AE. All patients should receive benzimidazoles, with very few exceptions. Only around 30% of AE‐patients can be cured (stage I to III a). For most patients, AE is a chronic disease with the need for long‐term drug therapy. As AE is rare, expertise is best acquired in a specialized institution; a benefit for the patients results from adherence to the WHO treatment recommendations. 48 Emerging Infections and Pneumonia P 20 Analysis of immune pathways and regulators to improve vaccine design M. Burggraf1,2, M. Riess1, Z. Melkova1, J. Seifried1, S. M. Yoh3, H. Yoh4, J. P. Ting4, S. K. Chanda3, K. Cichutek1,2 R. König1,3,2 1
Paul‐Ehrlich‐Institut, Host Pathogen Interactions, Langen, Germany Deutsches Zentrum für Infektionsforschung (DZIF), Langen, Germany 3
Sanford Burnham Prebys Medical Discovery Institute, Immunity and Pathogenesis Program, La Jolla, USA 4
Lineberger Comprehensive Cancer Center, Department of Microbiology and Immunology, Chapel Hill, USA 2
In recent years, the critical role of the innate immune system for the elicitation of an effective adaptive immune response has become an emerging concept. There had been a revolution in understanding of the receptors and molecules that drive innate immune responses in myeloid cells, such as dendritic cells (DCs). They play an essential role in linking innate and adaptive immunity as well as in the induction of vaccine‐
mediated immune responses. This project aims to understand the innate immune pathways and regulators affecting vaccine vector platforms in human dendritic cells that may constitute as a predictor of the immune responses in human individuals. Specifically relevant for early innate responses, differences in pattern recognition receptors and signaling pathways between humans and rodents may account for considerable differences. Therefore, it is of utmost importance to analyze a specific vaccine platform ‐ pathogen combination in a primary human cell system in vitro essential for the accelerated vaccine development at the TTU EI as a synergistic approach to guide future clinical trials. We are currently employing the ‐ at the TTU EI ‐ most progressed vector platforms for emergency vaccines, such as MVA and the Measles‐Virus platform, and compare their early innate signatures in a primary human cell model in vitro. Furthermore, we aim to identify novel regulators of the innate pathways, which can be targeted for improved vaccine design. We just recently identified one of the proximal receptors for pathogen DNA recognition, PQBP1, as part of the innate immune pathway in primary human dendritic cells (Yoh et al., Cell, 2015). Interestingly, MVA DNA recognition was not dependent on the PQBP1/cGAS complex. PQBP1 deficiency didn't lead to upregulation of an ISG signature. Furthermore, we identified a potent negative regulator of innate immunity, nucleotide‐binding, leucine‐rich‐repeat‐containing protein, NLRX1. We are currently evaluating the influence of NLRX1 deficiency in response to DNA viruses. These proteins are potential key players during initiation of an antiviral innate immune response and therefore potent vaccine targets. Interestingly, early innate immune responses happening within hours after vaccination have been shown to be a predictor for specific adaptive immune responses in humans occurring within days or weeks later. Therefore, our study will not only enhance our understanding of the mechanisms underlying the innate response, but also potentially analyze the correlates of protection of fast‐track vaccines in human. 49 Emerging Infections and Pneumonia P 21 Clostridium difficile infections in communities of Germany, Ghana, Indonesia, and Tanzania U. Groß1, P. Cooper2, K. Gunka1, I. Hasibuan1, I. Janssen1, R. L. Kusumawati3, V. Lang1, S. E. Mshana4 L. von Müller5, B. Okamo4, J. R. Ortlepp6, M. Rupnik7, W. Schneiderhan1, O. Zimmermann1, R. Daniel8 J. Overmann9 1
Universitätsmedizin Göttingen, Medizinische Mikrobiologie, Göttingen, Germany St. Martin de Porres Hospital, Eikwe, Ghana 3
University of Sumatra, Medan, Indonesia 4
Catholic University of Health and Allied Sciences, Mwanza, Tanzania 5
University of Saarland, Homburg/Saar, Germany 6
Asklepios Kliniken Schildautal, Seesen, Germany 7
University of Maribor, Maribor, Slovenia 8
University of Göttingen, Göttingen, Germany 9
DSMZ, Braunschweig, Germany 2
Problem Clostridium difficile infections (CDI) are considered as emerging nosocomial infections and may range from asymptomatic carrier status to mild diarrhea, eventually developing into pseudomembranous colitis up to a toxic megacolon that often results in high mortality. The use of antibiotics has been identified as a major risk factor for the development of CDI. We therefore undertook the effort to compare the epidemiology of CDI between countries in which the use of antibiotics is different. Methods A total of 594 healthy individuals and 608 patients suffering from diarrhea in communities in Germany, Ghana, Tanzania and Indonesia were selected for a cross‐sectional study. The study populations were screened for the presence of Clostridium difficile in stool samples. Cultured Clostridium difficile strains (n=97) were further subtyped and characterized using genome analysis, PCR‐ribotyping, MALDI‐TOF MS, determination of toxin production, and antibiotic susceptibility testing. Results Clostridium difficile was present in all countries, although with different prevalence rates in symptomatic patients: Germany (27%) > Indonesia (15%) > Africa (5‐6%). Differences were also obvious for genome and phage size, as well as ribotype distribution and toxin repertoires. Predominant ribotypes were 001/072 and 078 in Germany, SLO160 and 017 in Indonesia, and 084 in Africa. A high percentage of atoxigenic strains was found in Africa. Importantly, the isolates differed extremely in their antibiotic resistance pattern: In comparison to the other countries more isolates from German patients were resistant against moxifloxacin. Conclusion The presence of CDI has been demonstrated not only in Germany, but also in African and Asian countries indicating that CDI should also be considered as a cause of diarrhea in low‐ to middle‐income countries. This work was funded by the Federal State of Lower Saxony, Niedersächsisches Vorab (VWZN2889). 50 Emerging Infections and Pneumonia P 22 Unique immune signature of Ebola virus disease in humans P. Ruibal1,2,3,4, L. Oestereich1,3,4, A. Lüdtke1,2,3,4, B. Becker‐Ziaja1,3,4, M. Carroll5,4, N. Magassouba6, S. Günther1,3,4 C. Muñoz‐Fontela1,2,3,4 1
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany Heinrich‐Pette‐Institute, Hamburg, Germany 3
German Center for Infection Research, Hamburg, Germany 4
European Mobile Laboratory Consortium, Hamburg, Germany 5
Public Health England, Porton Down, Great Britain 6
Université Gamal Abdel Nasser, Conakry, Guinea 2
Despite the fact that we are still witnessing the largest Ebola virus disease (EVD) outbreak of all time, there is still a fundamental lack of knowledge about the pathophysiology of EVD. In particular, very little is known about human immune responses to Ebola virus (EBOV). In vitro studies and animal model research has pointed out to a strong immune suppression associated with EBOV infection, with involvement of inhibition of innate and adaptive immunity. However, whether this immunosuppression occurs in humans and the underlying mechanisms are not known. Here, we have for the first time evaluated the physiology of the human immune response in EVD patients from admission at the Ebola Treatment Center (ETC) in Guinea until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we have identified an immune signature that is unique in EVD fatalities and thus, predicts outcome. Fatal EVD is characterized by high expression of the T cell inhibitory molecule cytotoxic T‐lymphocyte‐
associated protein 4 (CTLA‐4) in CD8 and CD4 T cells, which was correlated with poor T cell activation and lack of viral clearance. In addition, CTLA‐4‐mediated internalization of immune stimulatory molecules from antigen‐
presenting cells (APCs) into CD8 T cells is presented as a possible mechanism for immune inhibition during EVD. To our knowledge, the identified T cell signature is the first immune‐based predictor of EVD, demonstrates a chief role of CD8 T cells for EVD survival, and points out to CTLA‐4 as a potential therapeutic target. Funded by European Union’s Horizon 2020 research and innovation program under grant agreement No 666100 (EVIDENT). 51 Emerging Infections and Pneumonia P 23 Ebola virus immunity in immunocompetent chimeric mouse models A. Lüdtke1,2, L. Oestereich2, P. Ruibal1,2, S. Günther2, C. Muñoz‐Fontela1,2 1
2
Heinrich Pette Institute, Emerging Viruses, Hamburg, Germany Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany Ebolaviruses are zoonotic RNA viruses from the Filoviridae family that cause severe disease in humans and non‐
human primates. Despite their importance as human pathogens very little is known about ebolavirus immune physiology. This is primarily due to the lack of immunocompetent small animal models of infection. To address this gap in the field, we engineered bone marrow chimeric mice via transplantation of wild‐type bone marrow cells into lethally irradiated type I interferon (IFN‐I)‐receptor knockout (IFNAR‐/‐) mice. Chimeric (IFNAR‐/‐)WT mice with restriction of IFN‐I deficiency to the stromal compartment supported productive EBOV infection but showed enhanced survival compared to the (IFNAR‐/‐)IFNAR‐/‐ control mice. Interestingly, (IFNAR‐/‐)WT mice exhibited low or no viremia revealing an important role of the hematopoietic‐driven immune response in controlling viral dissemination. In agreement with this, antibody‐based depletion experiments indicated that both CD8 and CD4 T cells were essentially required to control virus dissemination in (IFNAR‐/‐)WT mice. To compare species‐specific adaptive immunity on EBOV infection, we transplanted human hematopoietic stem cells into severely immunodeficient non‐obese diabetic (NOD)/severe combined immunodeficiency (scid)‐
interleukin‐2 (IL‐2) receptor‐g chain knockout (NSG) mice to generate ‘humanized’ mice. Infection of huNSG mice with non‐adapted EBOV was associated with viremia, cell damage, liver steatosis, signs of hemorrhage, and high lethality. Interestingly, this susceptibility to EVD was completely abolished in mice transplanted with mouse hematopoietic stem cells, indicating that the susceptibility of these mice to non‐adapted EBOV was completely dependent on the presence of human donor cells. Here we present two novel immunocompetent mouse models that are susceptible to non‐adapted EBOV and that allow species‐specific dissection of the mechanisms of EBOV immunity. Project Funded by DZIF/EBOCON. 52 Emerging Infections and Pneumonia P 24 Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein. A. Volz1, A. Kupke2, F. Song1, S. Jany1, R. Fux1, H. Shams‐Eldin 2, J. Schmidt2, C. Becker3, S. Becker2, G. Sutter1 1
LMU,Institute for Infectious Diseases and Zoonoses, Chair for Virology, Munich, Germany Philipps University Marburg, Institute of Virology, Marburg, Germany 3
University of Giessen, Lung Center, Department of Internal Medicine II, Section of Infectious Diseases, Giessen, Germany 2
Middle East Respiratory Syndrome coronavirus (MERS‐CoV), a novel infectious agent causing severe respiratory disease and deaths in humans, was first described in 2012. Modified Vaccinia virus Ankara (MVA), a highly attenuated strain of vaccinia virus originating from growth selection on chicken embryo fibroblasts serves as one of the most advanced recombinant poxvirus vectors in preclinical research and human clinical trials for developing new vaccines against infectious disease and cancer. Our objective was to use recombinant MVA compatible with clinical evaluation to express the MERS‐CoV spike (S) protein which is considered a key component of vaccines against coronavirus infections. The cDNA containing the entire gene sequence encoding the S antigen was obtained by DNA synthesis and modified by introducing silent mutations that remove three termination signals for vaccinia virus transcription. The target genes were cloned into MVA vector plasmids and introduced by homologous recombination into the MVA genome. The full‐length S protein of MERS‐CoV, expressed by MVA, is produced as ~210 kDa N‐glycosylated protein that is specifically recognized by antibodies in Western blot analysis. Further studies suggest cleavage of the mature full‐length S glycoprotein into an amino‐terminal domain (S1) and a ~85 kDa carboxy‐terminal domain (S2) that is putatively anchored to the membrane. When tested as a vaccine in BALB/c mice, recombinant MVA expressing the S protein (MVA‐MERS‐
S) were inoculated twice within a 21‐day interval. The intramuscular or subcutaneous immunizations with 106, 107 or 108pfu MVA‐MERS‐S vaccine induced circulating antibodies that efficiently neutralized MERS‐CoV infections in highly permissive tissue cultures. Transduction with recombinant adenovirus delivering the human receptor molecule DPP4 allowed assessment of protective capacity of vaccination upon respiratory challenge with MERS‐CoV. All mock vaccinated control mice developed high loads of MERS‐CoV in the lungs at day 5 post challenge. In contrast, MERS‐CoV replication was severely impaired in all animals immunized with MVA‐MERS‐S vaccines as demonstrated by more than 100‐fold reduced loads of MERS‐CoV. In conclusion, our study demonstrated the safety, immunogenicity and protective capacity of this MVA‐MERS‐S vaccine in a mouse model as precursor for future phase 1 clinical evaluation in humans. 53 Emerging Infections and Pneumonia P 25 Minimal cell surface factor requirements for filoviral cell entry N. Atenchong1, E. Dietzel2, M. Manns1,3, S. Ciesek4, S. Becker5, T. von Hahn1,6 1
Medizinische Hochschule Hannover, Klinik für Gastroenterologie, Hepatologie und Endokrinologie, Hannover, Germany Philipps‐Universität , Institut für Virologie, Marburg, Germany 3
Medizinische Hochschule, Klinik für Gastroenterologie, Hepatologie und End, Hannover, Germany 4
Medizinische Hochschule , Klinik für Gastroenterologie, Hepatologie und End, Hannover, Germany 5
Philipps‐Universität, Institut für Virologie, Marburg, Germany 6
Medizinische Hochschule , Klinik für Gastroenterologie, Hepatologie und End, Hannover, Germany 2
Background and aims Based on numerous individual studies a large number of molecules on the host cell surface has been reported to support filoviral cell entry. We aimed to comprehensively analyze the presence or absence of proposed cell surface filovirus entry factors (CSFEF) in filovirus cell entry on permissive and resistant cells in order to find out if minimal CSFEF requirements for permissiveness can be defined. Methods First, panels of cell lines either highly permissive or resistant to filovirus cell entry were assembled. Second, permissiveness and resistance was correlated to expression of potential CSFEFs based on microarray and protein expression data. Third, based on findings from the previous two steps key CSFEFs were overexpressed or silenced in resistant and permissive cells respectively. Lentiviruses pseudoparticles bearing filoviral glycoprotein (EBOVpp) were used for the majority of functional assays; key findings were validated using fully infectious Ebola virus (EBOV). Results In addition to known lymphocyte‐derived cell lines resistant to filoviral cell entry we identified two new EBOVpp and EBOV resistant cell lines of non‐lymphocytic origin. There was no difference between permissive and resistant cells in terms of expression of intracellular entry factors such as endosomal cathepsins or NPC1. While many permissive cell lines expressed somewhat higher CSFEF levels than resistant cells, there was no single CSFEF or combination of CSFEFs that reliably predicted filovirus permissiveness in all cases. Of note, we identified a highly permissive cell line that showed very low levels of all known CSFEFs. One of the known CSFEF, the Tyro3 kinase receptor Axl, significantly enhanced entry in one of the resistant cell lines without reaching the level of permissiveness seen in endogenously permissive cell lines. Moreover, shRNA‐mediated removal of Axl had no significant effect on filoviral cell entry in several other permissive cell lines suggesting that Axl is at least not essential. Conclusions Viral cell entry can determine cellular permissiveness and resistance to filovirus infection. The current knowledge of filovirus cell entry does not allow prediction whether a given cell will be permissive or resistant. The ubiquitous presence of known intracellular entry factors argues against a model where cell entry permissiveness is determined after non‐specific interactions at the cell surface have triggered uptake of the incoming virion. Moreover, known CSFEFs do not bestow full permissiveness to filovirus cell entry upon endogenously resistant cell lines. Additional as yet unknown CSFEF likely exist. 54 Emerging Infections and Pneumonia P 26 Temporal and spatial analysis of the 2014‐2015 Ebola virus outbreak in West Africa M. W. Carroll1,2,3, D. A. Matthews4, J. A. Hiscox5, M. J. Elmore2, A. Rambaut6,7,8, S. Diederich1,9,10, A. Kurth1,11 A. Lüdtke1,9,12, D. Muth1,9,13, T. Strecker1,9,14, J. Trautner1,15, R. Kerber16,1,9, R. Wölfel1,9,17, P. Formenty18 S. Günther 16,1,9 1
The European Mobile Laboratory Consortium, Hamburg, Germany Public Health England, Salisbury, Great Britain 3
University of Southampton, Southampton, Great Britain 4
University of Bristol, Bristol, Great Britain 5
University of Liverpool, Liverpool, Great Britain 6
University of Edinburgh, Institute of Evolutionary Biology, Edinburgh, Great Britain 7
National Institutes of Health, Bethesda, USA 8
University of Edinburgh, Centre for Immunology, Infection and Evolution, Edinburgh, Great Britain 9
German Centre for Infection Research (DZIF), Braunschweig, Germany 10
Friedrich Loeffler Institute, Greifswald, Insel Riems, Germany 11
Robert Koch Institute, Berlin, Germany 12
Heinrich Pette Institute, Hamburg, Germany 13
University of Bonn, Bonn, Germany 14
Philipps University Marburg, Marburg, Germany 15
Thünen Institute, Hamburg, Germany 16
Bernhard Nocht Institut for Tropical Medicine, Hamburg, Germany 17
Bundeswehr Institute of Microbiology, Munich, Germany 18
World Health Organization, Geneva, Switzerland 2
West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a twoyear‐old boy in December 2013. From this index case the virus was spread by human‐to‐human contact throughout Guinea,Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/ Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak. 55 Emerging Infections and Pneumonia P 27 Knowledge, attitude and practices of general practitioners regarding pneumococcal vaccination in Germany C. J. Klett‐Tammen1,2, G. Krause1,2, S. Castell1 1
2
Helmholtz Centre for Infection Research, Epidemiology, Braunschweig, Germany Hannover Medical School, Hannover, Germany Introduction Pneumococcal vaccination coverage in the elderly is low in Germany. Advice by general practitioners (GP) might influence the vaccination uptake in this age‐group. However, little is known about knowledge, attitudes, and practices (KAP) of German GPs regarding this vaccine. Objective The aim of our study was to explore GPs’ knowledge, attitudes, and practices connected with pneumococcal vaccination in the elderly to gain insight into possibilities for improvement of advice given by German GPs regarding pneumococcal vaccination. Materials and Methods A cross‐sectional study in GPs was conducted in Germany in spring 2015. 5000 practices were contacted by letter. The questionnaire consisted of 15 questions regarding vaccinations in the elderly, leading to 107 variables related to socio‐demographic and practice‐characteristics (12), (subjective) knowledge (2), attitudes towards vaccinations (67), and practices (26). We conducted a logistic regression, using backward selection with p Results 774 GPs (15.5%) participated, of which 99.3% reported to know the official STIKO recommendations and 93.4% to trust them. 16.1% of the GPs stated to provide vaccination consultation routinely at regular intervals; 80.4% always recommended the pneumococcal vaccine in the elderly if it was medically indicated, the main barrier was forgetting about it (70%). GPs in the eastern part of Germany are less likely to neglect this vaccination (OR: 0.46, 0.26‐0.82). No knowledge variables were significantly associated with recommendation of pneumococcal vaccination by GPs; while 4 attitude factors, like no trust in the official STIKO recommendations (OR: 15.39, 3.08‐76.80), were. For practice‐related variables, advising on vaccination routinely at regular intervals (OR: 0.28, 0.12‐0.67) and receiving information by professional journals (OR: 1.89, 1.12‐3.21) showed a significant association. Conclusion Less than one fifth of the participating GPs reports to advice about vaccinations at regular intervals. Only about 80% of GPs in our study report to have recommended pneumococcal vaccination always to elderly patients if medically indicated. Beyond the substantial impact of the place of residence, lack of trust in the official STIKO recommendations and providing routine vaccination consultation are strongly associated with advising elderly patients on the pneumococcal vaccine. Most GPs stated to trust the STIKO recommendations; main reason to not recommend pneumococcal vaccination was failure to remember. To enhance vaccine recommendations by GPs, it might be advisable to promote routine vaccination advice and an appropriate recall system which is integrated in practice‐software. 56 Emerging Infections and Pneumonia P 28 Macrophage released TNF‐related apoptosis‐inducing ligand (TRAIL) contributes to impaired alveolar fluid clearance after Influenza A virus infection C. Becker1, L. Morales‐Nebreda2, B. Selvakumar1, E. Lecuona2, I. Vadasz1, R. Morty1, C. Schmoldt1, T. Wolff3 S. Pleschka4, K. Mayer1, S. Gattenlöhner5, L. Fink6, J. Lohmeyer1, W. Seeger1, J. Sznajder2, G. Mutlu2 S. Budinger2, S. Herold1 1
University of Giessen Lung Center, Innere Med II, Giessen, Germany Northwestern University, Chicago, USA 3
RKI, Berlin, Germany 4
University of Giessen, Institut für Virologie, Giessen, Germany 5
University of Giessen, Department of Pathology, Giessen, Germany 6
Institute of Pathology and Cytology, Wetzlar, Germany 2
Rationale Influenza A viruses (IAV) can cause primary viral pneumonia resulting in acute respiratory distress syndrome (ARDS) associated with intensive edema formation. As impairment of edema resolution in ARDS patients is correlated with high mortality, we investigated changes in Na,K‐ATPase, a major regulator of fluid homeostasis, to define mechanisms affecting alveolar fluid clearance (AFC) in IAV‐infection. Methods The effects of IAV infection on Na,K‐ATPase have been studied in primary murine and human alveolar epithelial cells (AEC) in monoculture and in co‐culture with bone marrow‐derived (BMM) or alveolar macrophages (AM) and in murine IAV‐infection. Cells and mice were infected with A/PR/8/1934 and Na,K‐ATPase expression was analyzed by Western blot after pulldown of surface proteins and in flow cytometry. Effects on net AFC were determined after IAV infection in vivo. The underlying signaling mechanisms were identified by use of chemical inhibitors, genetic knockout approaches and by adoptive transfer of BMM in vivo, and were targeted by therapeutic application of neutralizing antibodies. Results Our studies demonstrated that surface expression levels of Na,K‐ATPase were significantly decreased after IAV‐
infection primarily in non‐infected cells in the monoculture and in presence of infected macrophages in the co‐
culture model, in response to a soluble mediator. We found type I interferons (IFN) and the macrophage released, IFN‐dependent cytokine TRAIL to be sufficient to decrease Na,K‐ATPase in a CaMKKβ‐ and AMPK‐
dependent way. Blockade of this pathway in vivo using trail‐/‐ or ifnar‐/‐ mice, or an adenovirally transduced dominant‐negative AMPK restored Na,K‐ATPase expression and AFC after IAV infection in vivo. Moreover, ccr2‐
/‐
mice lacking pulmonary macrophage recruitment showed improved AFC after IAV‐infection, whereas adoptive transfer of BMM derived from infected wt but not trail‐/‐ mice to ccr2‐/‐ decreased Na,K‐ATPase expression and AFC again. Targeting this pathway from a therapeutic standpoint, using neutralizing antibodies directed against type I interferons and TRAIL, significantly improved outcome. Conclusion Together, we demonstrate that AFC in IAV infection is impaired by reduced Na,K‐ATPase expression in AEC and that IFN‐induced BMM‐released TRAIL plays a critical role herein. Targeting the underlying molecular pathways might provide new treatments to improve fluid clearance and outcome in patients with IAV‐induced ARDS. 57 Emerging Infections and Pneumonia P 29 Lungenpestausbruch auf Madagaskar: Die Pest, eine immer noch gefürchtete Infektionskrankheit H. Scholz1, J. Riehm2 1
2
Institut für Mikrobiologie der Bundeswehr, Bakteriologie und Toxikologie, Munich, Germany Zentrales Institut des Sanitätsdienstes der Bundeswehr, Veterinärmedizin, Garching‐Hochbrück, Germany Die Infektionskrankheit und Zoonose Pest wird durch das Gram‐negative Bakterium Yersinia pestis verursacht. Kaum eine andere Erkrankung in der Geschichte der Menschheit hat so viel Angst und Schrecken verbreitet und Millionen von Menschenleben gekostet. Aber auch heute noch ist die Pest in zahlreichen Ländern Afrikas, Asiens und Amerikas endemisch. Naturherde der Pest persistieren insbesondere in Nagetierspezies, Insektenfressern, kleineren Raubtieren und deren Parasiten. Wie gefährlich die Pest auch heute noch sein kann, zeigen jüngste Ausbrüche von Beulen‐ und Lungenpest auf der Insel Madagaskar. Madagaskar zählt mit bis zu über 1000 humanen Pestfällen pro Jahr zu den Hochendemiegebieten der Pest. Unsere Studie analysiert einen Lungenpestausbruch im äußersten Norden der Insel, einem Gebiet, das bisher als frei von der Pest galt. Innerhalb von 27 Tagen wurden 2 Familien fast komplett ausgelöscht. Von insgesamt 22 potentiell Infizierten, verstarben 15 innerhalb weniger Wochen. Die Letalität bei unbehandelten Patienten lag dabei bei 100%. Aufgrund der weit abgelegen Lage des Ausbruchsgeschehens, erfolgte eine Antibiotikatherapie verzögert und eine Anzucht des Erregers war nicht möglich. Dennoch gelang es aus Patientenmaterial den Erreger mittels CRISPR (clustered regularly interspaced short palindromic repeat‐
Analysen) zu genotypisieren. Ebenfalls konnte Yersinia pestis in Nagern im unmittelbaren Ausbruchsbereich nachgewiesen und typisiert werden. Dies zeigt, dass auch in dieser Gegend der Pesterreger in Nagetieren persistiert. Unsere Untersuchungen lassen den Schluss zu, dass der Erreger Pest im Laufe der Geschichte nichts an seiner Gefährlichkeit / Virulenz eingebüßt hat. Eine rasche Therapie ist für das Überleben unbedingt erforderlich. Das Auftreten multiresistenter Peststämme auf Madagaskar könnte in Zukunft eine effektive Therapie erschweren. 58 Emerging Infections and Pneumonia P 30 Episodes of acute respiratory infection in toddlers ‐ a methodological comparison of the effect of episode definition on estimated disease burden in a prospective cohort study using symptom diaries B. Zoch1,2, A. Günther2, A. Karch3,2, R. T. Mikolajczyk3,2 1
PhD Programme “Epidemiology", Braunschweig‐Hannover, Germany Helmholtz Centre for Infection Research, ESME ‐ Epidemiological and Statistical Methods Research Group, Braunschweig, Germany 3
German Centre for Infection Research, Hannover‐Braunschweig site, Braunschweig, Germany 2
Background Acute respiratory infections (ARI) are among the most frequent childhood diseases in Western countries. Due to their mild nature, they rarely lead to contact with the healthcare system. As a consequence, definition of ARI episodes for research purposes needs to be based on medical history. This is usually assessed by retrospective surveys via parent‐administered questionnaires or by the application of parent‐administered prospective symptom diaries. However, definitions used for ARI episodes in research settings vary widely in the literature. It is the aim of our analysis to apply different episode definitions obtained from the literature to the same dataset from a prospective cohort study in Lower Saxony and to compare number and duration of ARI episodes captured by the respective definitions. Methods A systematic literature review was performed in order to identify ARI episode definitions used in research studies. The identified definitions were then applied to a symptom diary dataset from a cohort study of toddlers (1 to 3 years old) performed in the winter season 2013/2014. This study was a pilot study for LöwenKIDS, a multi‐centre population‐based birth cohort study focusing on the effect of infections and the human microbiome on the development of the immune system. LöwenKIDS started recruitment in 2015 and will include 500 children which follow an extensive data and biosample collection schedule up to the age of 6 years. Results Five ARI episode definitions were extracted from the literature; they differed with respect to the type and duration of symptoms and the symptom‐free interval necessary for the definition of a unique ARI episode. Dependent on the ARI definition used, the total number of identified episodes in our study varied from 144 to 231. The median number of episodes per child ranged from 1 to 3; the overall mean duration of ARI episodes in our dataset was 10 days when using the most restrictive episode definition and 15 days when using a more liberal one. Discussion Applying different literature definitions to the same cohort study dataset led to considerable differences in the number and duration of ARI episodes. Direct comparisons of study results obtained via different definitions should thus not be performed. We propose the use of a standardized ARI episode definition in upcoming cohort studies working with diary data as for example the LöwenKIDS birth cohort study. 59 Emerging Infections and Pneumonia P 31 Chimeric mice with competent hematopoietic immunity reproduce key features of severe Lassa Fever L. Oestereich1, A. Lüdtke2, T. Rieger1, S. Wurr1, E. Pallasch1, S. Bockholt1, C. Muñoz‐Fontela2,1, S. Guenther1 1
2
Bernhard Nocht Institute for Tropical Medicine, Virology, Hamburg, Germany Heinrich‐Pette‐Institut, Leibniz Institute For Experimental Virology, Hamburg, Germany Lassa fever (LASF) is a highly pathogenic viral syndrome endemic to west African countries. Despite the high morbidity and mortality caused by Lassa fever yearly, very little is known about the pathophysiology of the disease. One of the reasons that has precluded basic research on LASV has been the lack of relevant small animal models that reproduce human disease. Immunocompetent laboratory mice are resistant to infection with Lassa virus (LASV) and, to date, only immunodeficient mice have shown some degree of susceptibility to experimental infection. Since immunopathology is thought to be a key component of LASV pathogenesis, we sought to develop a susceptible mouse model with intact adaptive, hematopoietic‐driven immune response. Using transplantation of wild‐type bone marrow cells into lethally irradiated type I interferon receptor knockout mice (IFNAR‐/‐), we generated chimeric mice that reproduced the main features of severe Lassa fever in humans, including high lethality, T cell‐mediated immunopathology, and inflammation‐mediated organ damage. 60 Emerging Infections and Pneumonia P 32 Therapeutic effect of Favipiravir against Lassa fever in immunocompetent chimeric mice L. Oestereich1, A. Lüdtke2, T. Rieger1, P. Ruibal2,1, S. Wurr1, E. Pallasch1, S. Bockholt1, C. Muñoz‐Fontela2 S. Guenther1 1
2
Bernhard Nocht Institute for Tropical Medicine, Virology, Hamburg, Germany Heinrich‐Pette‐Institut, Leibniz Institute For Experimental Virology, Hamburg, Germany The pyrazinecarboxamide derivative T‐705 (favipiravir) was published in 2002 by Toyama Chemicals (Japan) as an inhibitor of influenza virus replication and is currently in clinical development for the treatment of highly pathogenic flu as well as Ebola virus disease. In this study, we tested the effect of favipiravir against Lassa fever in a recently generated immunocompetent mouse model of disease. Favipiravir presumably acts as a nucleotide analog that selectively inhibits the viral RNA‐dependent RNA polymerase or causes lethal mutagenesis upon incorporation into the virus RNA. Thus, besides influenza virus, favipiravir has shown potent antiviral activity against other segmented negative‐strand RNA viruses such as arena‐ and bunyaviruses both in vitro and in vivo. Following this rationale, we tested the therapeutic effect of favipiravir against Lassa fever (LASF), a severe febrile disease endemic to west African countries whose ethiologic agent is Lassa virus, a segmented, single stranded RNA virus. To model the disease in laboratory mice, we utilized recently generated immunocompetent models susceptible to the disease, in which stromal cells were IFNAR‐/‐ and the hematopoietic compartment was fully immunocompetent. Oral administration of favipiravir at 300 mg/kg after symptom onset (day 4 post‐infection) rescued 100% of infected mice, and resolved viremia in 48 h. Furthermore, half dose of favipiravir was sufficient to successfully rescue mice from LASF when administered in combination with Ribavirin, the drug of choice for current treatment of LASV. Our results point out to favipiravir as a highly promising candidate for treatment of LASF, and indicate a synergistic effect between favipiravir and ribavirin. 61 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 33 Identification of novel inhibitors targeting the type‐three secretion system of gastrointestinal pathogens S. Mühlen1, U. Bilitewski2, P. Dersch1 1
2
Helmholtz‐Zentrum für Infektionsforschung, Molekulare Infektionsbiologie, Braunschweig, Germany Helmholtz‐Zentrum für Infektionsforschung, Biologische Systemanalyse, Braunschweig, Germany A major part of the enteropathogenic bacterial species, such as pathogenic E. coli (EPEC, EHEC, EIEC etc), Salmonella, Shigella and Yersinia are members of the large family of Enterobacteriaceae. Most of these pathogenic gastrointestinal bacteria use a type‐three secretion system (T3SS) to translocate effector proteins directly into the host cell cytoplasm where they modify host cell signalling pathways in favour of bacterial survival and replication. Due to the fact that T3SSs are only expressed by pathogenic species but not the common gastrointestinal microbiota, T3SSs are an excellent target for therapeutics. Furthermore, as the secretion systems are highly conserved among the pathogenic species, it is highly likely to find inhibitors that may target not only one but all T3SS‐expressing gastrointestinal pathogens. We established a high‐throughput screen for the identification of natural and chemical compounds to use as specific inhibitors for the T3SS. Using β‐lactamase (BlaM) fusions to the known translocated effector protein Tir of EPEC, we can monitor the effect of the inhibitors on the translocation efficiency of the Tir‐BlaM fusion protein into Hep‐2 cells by staining with the FRET substrate CCF4‐AM. Cleavage of this substrate by β‐lactamase results in a change of fluorescence from green to blue indicating efficient translocation. We use a tir‐blaM fusion that has been reintroduced into the original genomic locus of tir as a tool. EPEC is known to express its T3SS under cell culture conditions (growth in DMEM). Therefore, we were able to set up two parallel screens inhibiting a) the formation of the T3SS by addition of the inhibitors during induction in DMEM and b) the contact of the T3SS with the host cell and formation of the pore in the host plasma membrane by addition of the inhibitors prior to infection of the cells. The high‐throughput screen of several available compound libraries has been completed and further characterisation of inhibitory compounds is currently underway. “Actives” are being tested for their cytotoxic effect on both bacteria and cells before moving on to determining their effect on the T3SSs of other pathogens such as Salmonella and Yersinia and further characterisation of the inhibitors. 62 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 34 New Delhi Metallo‐beta‐Lactamase 1 substrate binding site mutant K211E: substrate and Zn2+ dependent activity H. Meyer1, C. Daschkin1, R. Nitiu1, D. Scheikl2, M. Gerhard1 1
2
Technische Universität München, Institut für medizinische Mikrobiologie, Immunologie und Hygiene, Munich, Germany Technische Universität München, Center of Life and Food Sciences Weihenstephan, Freising, Germany Background Being discovered in 2009, the New Delhi Metallo‐beta‐Lactamase 1 (NDM‐1) has reached immediate attention due to its exceptionally broad substrate promiscuity conferring resistance to all beta‐lactam antibiotics and its rapid dissemination both across the globe as well as between bacterial species. Moreover, being frequently plasmid encoded in combination with various resistances against most other classes of antibiotics, NDM‐1 positive bacterial pathogens are extremely difficult to treat and pose an enormous threat to human health. Inhibition of the NDM‐1 activity is expected to broaden the treatment options against NDM‐1 positive pathogens and thereby meet this growing medical need. However, this approach is impeded as no Metallo‐
beta‐Lactamase inhibitors are in clinical development or use so far and as the development of NDM‐1 specific inhibitors is hampered by the exceptionally large active site. For obtaining structural information about the required active site geometry we recombinantly produced both wild type and K211E substrate binding site mutant and compared their enzymatic activities using different beta‐lactam substrates at various Zn2+ concentrations. Methods NDM‐1 WT and K211E mutant were recombinantly produced using a SUMO tag / SUMO protease system. The activity of both NDM‐1 forms was monitored at various Zn2+ concentrations in a colorimetric assay in which the hydrolysis of the structurally related substrates CENTA and Nitrocefin (Cephalosporins) as well as Imipenem and Meropenem (clinically relevant Carbapenems) was monitored. Moreover, the inhibitory effect of Captopril, a published NDM‐1 inhibitor, was determined with different substrates and Zn2+ concentration. Results The absolute and relative activity of WT versus K211E mutant NDM‐1 differ substantially depending on the type of substrate and Zn2+ concentration used in the biochemical assay. Even within the Cephem class of beta‐
Lactam substrates the WT enzyme shows clear differences in the specific activity, with CENTA being a more efficient substrate than Nitrocefin. In contrast, the K211E mutant, which is completely inactive with Imipenem or Meropenem as substrate, shows limited activity with CENTA, but exceeds WT activity with Nitrocefin at increasing Zn2+ concentrations. Moreover, the inhibitory effect of Captopril, a published inhibitor of NDM‐1, clearly depends both on the substrate and Zn2+ concentration used. Conclusions Enzymatic activity of both NDM‐1 forms and the inhibitory effect of Captopril markedly depend on the type of substrate and the Zn2+ concentrations used. This has strong implications on screening procedures for NMD‐1 inhibitors for clinical use. Besides using clinically relevant beta‐Lactams as substrates, physiological Zn2+ concentrations have to be considered when searching for NDM‐1 inhibitors using biochemical b‐Lactam hydrolysation assays. 63 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 35 Preclinical C57BL/6 mouse model of Pseudomonas aeruginosa colonization and infection in the immunocompromised host F. Hölzl1, H. Hinkov1, M. Willmann1, S. Peter1, K. Gronbach1, I. Autenrieth1, J.‐ S. Frick1 1
Institut für Med. Mikrobiologie und Hygiene, AG Frick, Tübingen, Germany The gastrointestinal tract contains the largest and most complex bacterial community in the human body, which protects its host from colonization and infection by both pathogenic and opportunistic organisms in concert with the immune system. Especially patients currently under immunosuppression are constantly threatened by nosocomial infections, including multi‐resistant pathogens such as extensively drug‐resistant (XDR) Pseudomonas aeruginosa. Colonization with XDR Pseudomonas aeruginosa is favored by prophylactic broad spectrum antibiotic therapy, which disrupts the microbiota‐mediated colonization and infection resistance. It is as yet unclear whether immunodeficiency, mucositis, an altered microbial pattern, the specific virulence/fitness factors of the pathogens themselves or a combination of these factors account for the increased susceptibility towards colonization and even bloodstream infection specifically with germs such as XDR Pseudomonas aeruginosa. This project aims to clarify the role of the immune system as well as the microbiota using a C57BL/6 mouse model for preclinical evaluation of interventions targeting the GI microbiota. Conventional and germfree C57BL/6 mice are orally exposed to Pseudomonas aeruginosa, resulting in a stable intestinal colonization. Along with a reference strain, XDR strains isolated from hemato‐oncological patients suffering from septicemia are employed. Immunodeficiency and alterations in the microbiota are induced through cyclophosphamide injection and/or antibiotic treatment. Fecal transplantation manages to suppress the colonizing germ and appears to temporarily restore colonization resistance, pointing towards the important protective function of the microbiota or of particular strains contained therein. Longitudinal changes in the microbial pattern are analyzed before and after treatment/exposure and the animals’ susceptibility to colonization and infection is evaluated. Sequencing of the microbiota allows for the selection of bacterial candidates for further intervention trials to try and suppress or possibly clear the problematic colonizing germs, as a potential prevention/therapy method for colonization or infection. 64 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 36 Multi‐resistant bacteria as cause for spontaneous bacterial peritonitis in patients with liver cirrhosis are rare in the setting of a German university hospital. P. Lutz1,2, H. D. Nischalke1,2, B. Krämer1,2, B. Langhans1, F. Goeser1,2, D. Kaczmarek1,2, M. Parcina2,3 J. Nattermann1,2, A. Hörauf2,3, C. P. Strassburg1,2, U. Spengler1,2 1
Universität Bonn, Medizinische Klinik I, Bonn, Germany DZIF, Bonn, Germany 3
Institut für Medizinische Mikrobiologie, Immunologie und Parasitologie, Bonn, Germany 2
Background and Aims Spontaneous bacterial peritonitis (SBP) is a life‐threatening infection in patients with liver cirrhosis, originating mainly from intestinal bacterial translocation. The standard treatment consists of third generation cephalosporins. However, clinical studies indicate that this treatment has become ineffective in particular in patients with nosocomial SBP. In settings with high prevalence of multi‐resistant bacteria, meropenem plus daptomycin are recommended as result of a randomized trial. We analysed antibiotic resistance in a cohort of patients from a tertiary medical centre to determine which empirical treatment may be appropriate in Germany. Methods All cases of SBP occurring from March 2012 till July 2015 in the Department of Internal Medicine I at the University of Bonn were analysed for the prevalence of antibiotic resistance among the isolated bacteria. According to international guidelines, SBP was diagnosed in patients with ascites due to liver cirrhosis if ascites PMN count exceeded 250 cells/µl in the absence of a contiguous source of infection. Results In total, we observed 86 episodes of SBP in 80 patients. Most patients were male (63%), had cirrhosis due to alcohol abuse (59%) and were classified as Child‐Pugh stage C (74%) as indicator of advanced liver disease. Median age was 59 years. As usual for SBP, the causative microorganism could be identified only in 54% of cases. SBP was nosocomial in 64%. The microorganisms isolated in non‐nosocomial and nosocomial SBP were comparable, with Escherichia coli (both 26%), streptococci (21% and 15%), Klebsiella species (16% and 15%) and enterococci (16% and 15%) being the most frequent. No multi‐resistant microorganism was detected. Resistance to antibiotics was found more often in nosocomial than in non‐nosocomial SBP: for quinolones in 50% versus 11% (p=0.009), for cephalosporins in 31% versus 26% (p=1.0), and for ureidopenicillins plus beta‐
lactamase inhibitor in 31% versus 11% (p=0.2). Resistance to penems was only detected in nosocomial SBP in 3 isolates of Enterococcus faecium, which were all sensitive to vancomycin. Conclusion Multiresistant bacteria seem to be rare as cause for SBP in the setting of a German university hospital. From the analysis of our cohort, the combination of meropenem plus vancomycin should be very effective for initial treatment of nosocomial SBP. 65 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 37 MicroTrans Registry for fecal microbiota transplantation in Germany ‐ an interim analysis S. Hagel1,2, A. Fischer1, D. Tacke3,4, O. A. Cornely3,4,5, A. W. Lohse6, M. Lerch7, A. Stallmach1, M. J. G. Vehreschild3,4 O. B. O. on behalf of the MicroTrans Study Group8 1
Jena University Hospital, Department of Internal Medicine IV (Gastroenterology, Hepatology and Infectious Diseases), Jena, Germany 2
Jena University Hospital, Integrated Research and Treatment Center, Center for Sepsis Control and Care (CSCC), Jena, Germany 3
University Hospital of Cologne, 1st Department of Internal Medicine and Center for Integrated Oncology CIO Cologne/Bonn, Cologne, Germany 4
German Centre for Infection Research (DZIF), partner site Bonn‐Cologne, Germany 5
Cologne Excellence Cluster on Cellular Stress Responses in Aging‐Associated Diseases (CECAD), and Clinical Trials Center Cologne, ZKS Cologne, BMBF 01KN1106, University of Cologne, Cologne, Germany 6
University Hospital Hamburg‐Eppendorf, 1st Department of Internal Medicine, Hamburg, Germany 7
Department of Internal Medicine A, University Hospital Greifswald, Germany 8
on behalf of the, MicroTrans Study Group, Germany Introduction Fecal microbiota transplantation (FMT) has become an increasingly applied individual treatment option for patients with recurrent infections caused by the bacterium Clostridium difficile (CDI). A recently published randomised clinical trial assessing FMT highlighted its impressive clinical efficacy of over 90% in achieving sustained clinical cure. However, the lack of standard operating procedures regarding donor screening, transplant preparation, and route of administration as well as the lack of an official regulatory classification of FMT is proving to be a major obstacle to this aim. Furthermore, the possible implications of allogeneic FMT are poorly understood and there are no long‐term data available. Materials and Methods A retrospective, multicenter study was initiated. Documentation comprises age, gender, height, weight, underlying disease, ECOG‐status, indication for FMT, antibiotic therapy and chemotherapy in the pre‐transplant period, immunosuppression pre‐transplant, bowel‐movement pre/post‐transplant, pre‐transplant analyses, details regarding the transplant preparation and transplant, administration, details regarding transplant‐donor, outcome, long‐term‐follow‐up (10 d, 4 weeks, 6, 12 and 24 months after FMT). Data is entered into a paper CRF or alternatively into an eCRF atwww.ClinicalSurveys.net. A positive vote of the institutional review board and/or ethics committee was obtained at all sites. Results So far, 35 centers performing FMT in Germany could be identified. A total of 100 FMT could be documented (range: 1‐12 per center). Median age was 58.6 years (range: 9‐108 years). 64 patients (64.0%) were female. 24 patients (24.0%) had received chemotherapy or immunosuppression in the 3 months prior to FMT. The procedure was performed after a median of 2.9 relapses of CDI (range: 1‐10). Vancomycin was given most frequently as a conditioning antibiotic regimen before FMT (n=48; 48.0%). Sixty‐two patients (62.0%) received a bowel lavage before FMT. Administration into the upper gastrointestinal tract by was most frequently practiced (n=52; 52.0%), followed colonoscopic administration (n=43; 43%) and capsules (n=5; 5.0%). Nausea (n=4; 4.0%), transient fever (n=4; 4.0%) and eructations (n=2; 2.0%) were the most commonly observed adverse events. None of the patients died as a consequence of an adverse event considered related to FMT. Response to treatment was 80%. Discussion FMT is an increasingly, yet heterogeneously practiced treatment regimen in Germany. Further documentation of real‐life treatment courses is warranted to develop standard operating procedures and reliably capture potential safety issues. 66 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 38 Identification of small‐molecule inhibitors targeting Cytotoxin‐associated gene (Cag) type IV secretion in Helicobacter pylori F. Schindele1, U. Bilitewski2, W. Fischer1, R. Haas1 1
2
Max von Pettenkofer‐Institut für Hygiene und Medizinische Mikrobiologie, Munich, Germany Helmholtz Zentrum für Infektionsforschung, Braunschweig, Germany The chronic gastric pathogen Helicobacter pylori infects approximately half of the world population and is estimated to cause at least 500,000 new cases of gastric cancer per year. H. pylori uses a type IV secretion system encoded on the cag pathogenicity island to inject the oncoprotein CagA into host cells ‐ a key event in the induction of cancer by H. pylori. As H. pylori becomes increasingly resistant to the current therapeutic antibiotics, alternative strategies targeting virulence factors without directly killing bacterial cells is a promising issue. We aim to identify small‐molecule inhibitors (SMI) against bacterial type IV secretion systems (T4SS), using the cag‐T4SS of H. pylori as a model system. We have engineered and optimized a suitable in vitro screening system for CagA translocation into gastric cells under high‐throughput conditions. This novel reporter system is based on a fusion of a TEM‐1 β‐lactamase to full‐length CagA. TEM‐1‐CagA translocation into target cells is monitored by TEM‐1‐mediated conversion of the fluorescent β‐lactam derivative CCF4. Using a 384‐well fluorescence plate reader format, we have performed medium‐throughput screenings of different libraries including natural products, synthetic compounds as well as already described pharmaceuticals. This screening procedure resulted in preliminary identification of various compound classes that were able to inhibit CagA translocation. The establishment of follow‐up protocols enabled us to identify whether these compounds modify the eukaryotic cell or directly target the bacterium leading to an inhibition of CagA translocation. Apart from substances with described antibacterial activity or some that have known targets of cellular origin, we have identified a set of new specific anti‐H. pylori compounds with IC50 values (CagA translocation) and MIC values (H. pylori growth) in the nanomolar range. Screening of further libraries is planned to optimize the output of suitable SMI candidates. Finally, established inhibitors that target the cag‐T4SS hold the potential not only to block the T4SS translocation of effectors in other pathogenic bacteria, but also to decrease the spread of antibiotic resistance. 67 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 39 The influence of host serum factors on the development of S. mansoni in its definite host S. Frahm1, E. Loffredo‐Verde1, S. Bhattacharjee1, S. Luo2, L. Formichella2, J. Keiser3, A. Verschoor2 C. Prazeres da Costa1 1
Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Parasitologie, Munich, Germany Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Munich, Germany 3
Schweizerisches Tropen‐ und Public Health‐Institut, Helminth Drug Development, Basel, Switzerland 2
Schistosomes are unique among the helminths since adult worms live in the blood vessels of their definite host where they are exposed to the full scale of cellular and humoral immune attacks. Nevertheless, they survive under such hostile conditions i.e. potentially harmful serum factors for up to ten years. This demonstrates they have developed strong and broad immune evasion mechanisms during co‐evolution which step into place during all stages of their development. The main focus of this work was to understand in detail how and when the resistance of S. mansoni against host serum factors arises. We therefore employed an established in vitro assay in which cercariae are mechanically transformed into and cultivated as skin and lung schistosomulae (also called “newly transformed schistosomulae” (NTS)). Up to now, the viability of NTS depended on media containing fetal‐calf serum (FCS) and therefore serum factors. We advanced this system to serum‐free condition allowing us to add different host‐sera and serum components under well‐defined conditions. We detected drastic differences in the viability of NTS when incubated with sera of different species. Whereas human serum propagated NTS maturation up to gut‐developing worm stage (day 18 onwards), NTS incubated with media containing FCS developed only to the lung migratory stage. In contrast serum of in‐bred C57BL/6 and outbred NMRI mice efficiently killed the NTS within 5 days. Further, addition of mouse serum into survival‐
propagating conditions also induced NTS‐killing which could point towards the presence of active component(s) within mouse serum. With sera from other species we could observe either the improved maturation (i.e. swine) or killing phenotype (i.e. rhesus monkey) in different degrees. Finally, we investigated the influence of the complement factor C1q as this was previously indicated to be inactivated by schistosomes. However, mouse serum deficient of C1q did not alleviate the NTS killing process and no influence was observed in the development of adult worms in S. mansoni infected C1q‐deficient animals. Our current studies focus on defining host‐specific serum factors responsible for either survival or killing of the NTS as well as on the role of other complement factors such as C3, C4 and C5 on the development of NTS in vitro. 68 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 40 Type I interferons modulate autophagy during Salmonella Typhimurium infection of macrophages N. Hos1, R. Ganesan1, S. Gutiérrez1, J. Stepek1, J. Fischer2, M. Krönke1, N. Robinson1 1
2
Uniklinik Köln, Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Cologne, Germany Uniklinik Köln, Klinik I für Innere Medizin, Cologne, Germany Introduction Type I interferons (IFN‐I) have been shown to be critical for antiviral immunity. However, their role upon bacterial infection largely remains unknown. We have recently demonstrated that Salmonella Typhimurium induces cell death in macrophages by the production of IFN‐I, which is accompanied by an increased number of autophagic vacuoles. Therefore IFN‐I might also regulate autophagy‐dependent targeting and degradation of S. Typhimurium in macrophages by a yet undefined mechanism. Objective In the present study we investigated the impact of IFN‐I on the molecular regulation of autophagy during S. Typhimurium infection of macrophages. Materials & Methods −/−
Bone marrow from wild‐type (WT) and IFN‐I receptor deficient (ifnar ) mice was cultured in RPMI medium containing 10% fetal bovine serum and was differentiated into macrophages for 7 days using macrophage colony‐stimulating factor (20 ng/mL). Bone marrow derived macrophages (BMDMs) were infected with S. Typhimurium (SL1344) at a multiplicity of infection (MOI) of 10 and samples were collected for whole transcriptome analysis using next‐generation sequencing, quantitative real‐time PCR (qRT‐PCR), Western Blot analysis and immunocytochemistry. Results Whole‐transcriptome analysis after 2 hr of S. Typhimurium infection revealed that major autophagic and lysosomal genes were downregulated in WT and ifnar −/− BMDMs. By using qRT‐PCR, we confirmed that mRNA levels of the transcription factor tfeb, which induces autophagy and the expression of important host defence genes, were also downregulated. We found that S. Typhimurium infection activated the metabolic checkpoint kinase mTOR in both WT and ifnar −/− BMDMs. Activated mTOR retained TFEB in an inactive form in the cytosol. Consistently, autophagy was inhibited after 2 hr of S. Typhimurium infection as indicated by the accumulation of the autophagy marker proteins LC3 II and p62. In ifnar −/− BMDMs, however, we observed a remarkable decrease in LC3 II after 4 and 8 hrs of infection. Conclusion S. Typhimurium infection leads to the reprogramming of autophagic and lysosomal pathways in macrophages. Upon S. Typhimurium infection, IFN‐I may play an important role in the regulation of autophagy and might therefore be a future target for combatting intracellular bacterial infections. 69 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 41 Imported or endemic? Sources of the large Vibrio cholerae outbreak in Ghana, 2014 D. Eibach1, S. Herrera‐Léon2, H. Gil2,3, B. Hogan1,4, L. Ehlkes1,4, M. Adjabeng5, B. Kreuels6, D. Opare5, J. Fobil7 J. May1,4 1
Bernhard‐Nocht‐Institut für Tropenmedizin, Hamburg, Germany Institute of Health Carlos III, Madrid, Spain 3
European Public Health Microbiology Programme (EUPHEM), European Centre for Disease Prevention and Control, Stockholm, Sweden 4
German Center for Infection Research (DZIF), partner site Hamburg‐Borstel‐Lübeck, Germany 5
Ghana Health Service, Accra, Ghana 6
University Medical Centre Hamburg‐Eppendorf, Hamburg, Germany 7
University of Ghana, Accra, Ghana 2
Introduction With more than 20,000 people affected in the year 2014, Ghana experienced one of its largest cholera outbreaks in more than a decade. To detect a causative newly imported strain or simultaneous outbreaks involving multiclonal strains, outbreak isolates were characterized, subtyped and compared to previous epidemics in 2011 and 2012. Methods For 92 Vibrio cholerae isolates from the years 2011, 2012 and 2014 the serotype, biotype, antibiotic susceptibility and the presence of ctxAB were determined. For a subgroup of 45 isolates pulsed‐field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST) and multilocus‐variable tandem repeat analysis (MLVA) was performed. Results 89 isolates (97%) were identified as ctxB (classical type) positive V. cholerae O1 biotype El Tor, with 88 strains belonging to serotype Ogawa and one strain belonging to serotype Inaba. Three (3%) isolates were cholera toxin negative non‐O1/non‐O139 V. cholerae. While only sulfamethoxazole/trimethoprim resistance was detectable in 2011, 95% of all 2014 strains showed multidrug resistance towards sulfamethoxazole/trimethoprim, ampicillin and ciprofloxacin. All subtyped O1 strains belonged to MLST sequence type 69. PFGE analysis revealed 11 pulsotypes with two main clusters, which could be further distinguished by MLVA into 22 genotypes and 3 clonal complexes (CC). The majority of isolates cluster by the outbreak period. Apart from those outbreak clusters additional genetically non‐related genotypes circulated during each annual epidemic. Conclusion This analysis suggests different endemic reservoirs of V. cholerae in Ghana with distinct annual outbreak clusters accompanied by the occurrence of genetically distant genotypes. Preventive measures for cholera transmission should focus on aquatic reservoirs and rapidly emerging multidrug resistance must be monitored closely. 70 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 42 Influence of host factors on Shiga Toxin 2 expression by Enterohemorrhagic Escherichia coli (EHEC) T. Baumgartner1,2, J. Glaser1,2, R. Gerlach3, B. Stecher1,2, M. Koeppel1,2 1
Max von Pettenkofer‐Institut, LMU, Munich, Germany German Center for Infection Research (DZIF), Partner site LMU Munich, Munich, Germany 3
Robert Koch‐Institut, Wernigerode Branch, Wernigerode, Germany 2
Enterohemorrhagic Escherichia coli (EHEC) are intestinal pathogens that can cause hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. These severe diseases are linked to the expression of Shiga toxins (Stx) which are encoded on prophages integrated in the bacterial genome (stx1/stx2). Stx expression is tightly regulated and closely linked to bacterial SOS‐response and prophage induction. Not all EHEC infected patients develop hemorrhagic colitis and only some progress to an HUS. The risk factors for developing HUS remain unclear as well as the signals triggering Stx production in the human gut. Our study aims to identify risk factors for the development of HUS in infected patients. In vitro experiments have shown that polymorphonuclear leukocytes (PMN) and reactive oxygen species (ROS) have an influence on Stx‐production (Wagner et al. Infection Immunity 2001). Since data on this topic is scarce, the aim of our study is to validate these findings and investigate the effect of other host‐derived factors on stx2 expression EHEC in vitro. In the next step, in vitro findings shall be tested in vivo (EHEC mouse infection model). To study stx2 expression in response to various host‐derived stimuli, we use a set of well characterized stx2 transcriptional reporter strains generated in our laboratory. The reporters carry genes for Gaussia luciferase (gluc) and gfp inserted into the stx2 locus, thereby rendering the EHEC strain BSL2. Human PMNs were isolated using a density gradient protocol. In addition we established quantification assays for ROS production by PMNs using fluorescent dyes. We have developed a medium‐throughput assay to detect luciferase activity upon stimulation and co‐culture with PMNs. Using this assay, we did not observe a robust activation of stx2 expression by PMNs, despite expression was activated by H2O2, the major ROS produced by PMNs. Quantification of H2O2 released by activated PNMs suggested that this concentration range is insufficient for stx2 induction. The role of PNMs in the Stx2 production by EHEC remains unclear. Therefore, we plan to study the effect of phagocytosis of the bacteria on stx2 expression using the gfp‐reporter strains. Further experiments will also focus on the role of other host factors such as lysozyme, the complement system and antimicrobial peptides. Wagner, P.L. el al. (2001). Human Neutrophils and Their Products Induce Shiga Toxin Production by Enterohemorrhagic Escherichia coli. Infect. Immun. 69, 1934‐1937. 71 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 43 Microbiota‐defined and clinical risk factors for Clostridium difficile colonization and ‐
associated disease in patient groups at risk: preliminary report on the multicenter, prospective SPECTRUM study P. Solbach1, M. J. G. Vehreschild2, N. Jazmati3, E. Tacconelli4, M. Buhl5, B. Schulte5, M. Gerhard6, C. Thoeringer7 A. Koch8, M. Manns1, J. Rupp9, C. Sina10, S. Suerbaum11, O. Bachmann1 1
Hannover Medical School, Dept. of Gastroenterology, Hepatology and Endocrinology, Hannover, Germany University of Cologne, 1st Department of Internal Medicine, Cologne, Germany 3
University of Cologne, Institute for Medical Microbiology, Immunology and Hygiene, Cologne, Germany 4
Tübingen University Hospital, Division of Infectious Diseases, Department of Internal Medicine 1, Tübingen, Germany 5
Tübingen University Hospital, Department of Clinical Microbiology and Hygiene, Tübingen, Germany 6
Technische Universität München, Institute for Medical Microbiology, Immunology and Hygiene, Munich, Germany 7
Technische Universität München, II. Medizinische Klinik und Poliklinik, Klinikum rechts der Isar, Munich, Germany 8
Hannover Medical School, Institute for Biometry, Hannover, Germany 9
University Hospital Schleswig‐Holstein, Department of Medical Microbiology and Hygiene, Lübeck, Germany 10
University Hospital Schleswig‐Holstein, Medical Department I, Lübeck, Germany 11
Hannover Medical School, Institute of Medical Microbiology and Hospital Epidemiology, Hannover, Germany 2
Background Clostridium difficile (C. diff.) is the major known cause of hospital‐acquired diarrhea. In particular, the increasing incidence over the last decades and the high recurrence rates after antibiotic treatment add to the significant clinical and economic burden. Microbial disruption is considered a key factor in the pathogenesis of C. diff. infection, but data on the specific components of the gut microbiota which are relevant for C. diff. colonization or CDI is sparse and mostly derived from cross‐sectional studies. Aim The aim of the ongoing SPECTRUM study (DRKS00005335) is thus to prospectively identify microbiota‐defined and clinical risk factors for C. diff. colonization and CDI in patient groups at risk. Methods 1100 patients belonging to pre‐defined risk groups were recruited within 48h of hospital admission to one of the 5 study sites (Hannover, Cologne, Tübingen, Munich and Lübeck). Stool samples were collected on admission, in case of CDI diagnosis, and at discharge, and screened for C. diff. using glutamate dehydrogenase testing (GDH EIA, Techlab, Blacksburg, USA). Results 704 patients provided >1 fecal sample and were thus eligible for further analysis. Clinical data and GDH test results were available for 281 and 576 patients, respectively. Average length of hospital stay was 7.84±9.18 days. Previous or concurrent treatment included antibiotics in 35.2%, and gastric acid suppressive agents in 91.5% of patients. GDH test was positive on admission and at discharge in 7.73%, on admission only in 5.21%, and at discharge only in 2.26%, while clinical CDI in combination with GDH positivity occurred in 1%. 255 patients provided only one fecal sample on admission with a positivity of 8.24%. Summary and Conclusion This interim report provides a preliminary characterization of clinical risk factors and C. diff. frequency in a prospective, longitudinal, multicentric approach studying patients at risk. Future PCR analysis of intestinal microbiota using the collected fecal samples is expected to generate a solid database on the protective and susceptible microbial taxa and may ultimately lead to the identification of probiotic agents for prevention and treatment of CDI. 72 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 44 A biobank of Giardia duodenalis isolates for functional epidemiology C. Klotz1, P. Gosten‐Heinrich1, T. Aebischer1 1
Robert Koch‐Institut, Fachgebiet 16, Berlin, Germany Gastro‐intestinal infections with protozoan parasites of the genus Giardia duodenalis are a worldwide endemic and represent a significant problem for Public Health. Treatment options are limited to a few approved drug classes. Up to 20% of giardiasis cases are treatment refractory to common therapy with nitroimidazoles and highlight the need of alternative treatment options. Pathogenesis and underlying virulence factors of the giardiasis are largely unknown. This project aims at the generation of a biobank and data base of G. duodenalis isolates from patients as a tool for functional epidemiology. Trophozoite cell lines are established from purified G. duodenalis cysts by in vitro excystation and culture protocols. The parasites are genetically characterized by analyzing the genomic sequence of established marker genes. The established trophozoite cell lines are characterized in functional assays regarding drug susceptibility or potential virulence factors. Currently, 264 cysts samples were subjected to in vitro excystation and long‐term trophozoite cultures could be established for 33 isolates (12.5%). Genetic characterization of the cysts revealed G. duodenalis assemblage type B in approximately 80% of the samples. In contrast, approximately 50% of the trophozoites from the established long‐term cultures belonged to assemblage A. Selected isolates have been characterized in more detail in vitro for their drug susceptibility to metronidazole and orlistat, a lipase inhibitor with potent Giardia growth inhibitory effects in vitro. The susceptibility to metronidazole and orlistat determined as IC50 ranged between values of 5‐13 µM and 1‐7 µM, respectively, depending on the parasite isolate type. In conclusion, the long‐term in vitro growth of 33 new G. duodenalis isolates constitutes a starting point for functional analysis. In particular, drug susceptibility and the diversity of potential virulence factors will be analysed in future in more detail to determine underlying genetic traits by correlative analysis. 73 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 45 CMV‐assoziiertes Ulcus ventriculi bei immunkompetentem Patienten ‐ ein Fallbericht U. Kastenbauer1 1
Praxis Dr. Ulrich Kastenbauer, Munich, Germany Wir berichten von einem 45‐jährigen immunkompetenten Patienten, der sich mit akuten Oberbauchschmerzen und Erbrechen vorstellte. In der Abklärung zeigte sich eine frische CMV‐Infektion als (Mit‐)Ursache eines floriden Ulcus ventriculi. Begleitend bestand ein passagerer zellulärer Immundefekt, der sich laborchemisch in einer Inversion der T4/T8‐Ratio, klinisch in einer Soor‐Ösophagitis manifestierte. Organmanifestationen im Bereich des oberen Gastrointestinaltraktss im Rahmen einer CMV‐Erstinfektion bei immunkompetenten Erwachsenen sind selten. In diesem Fall kam es zur kompletten Abheilung des Ulcus unter Therapie mit Protonenpumpeninhibitor, eine virostatische CMV‐Therapie war nicht notwendig. 74 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 46 Role of intradomain flexibility and dissociation propensity of the C‐terminal domain of SpaS in the mechanism of substrate specificity switching of bacterial type III secretion systems M. Bachmann1, J. Monjarás Feria1, S. Wagner1 1
Universität Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, Tübingen, Germany Type III secretion systems (T3SS) are used by many pathogenic or symbiontic Gram negative bacteria to inject effector proteins into eukaryotic host cells in order to promote bacterial survival and colonization. Salmonella enterica serovar Typhimurium (S. Typhimurium) encodes one of these systems within its pathogenicity island 1 (SPI‐1). It is essential for this pathogen’s ability to invade and replicate within mammalian host cells. T3SSs are among the most complex protein secretion systems known. The core unit of the systems is a cell envelope‐spanning macromolecular machine termed needle complex. It consists of a base that anchors the complex in the bacterial inner and outer membranes, a cytoplasmic apparatus involved in targeting and preparation of substrates, an inner membrane‐embedded export apparatus, and the filamentous inner rod, needle, and needle tip that protrude from the bacterial surface and serve as conduit for substrates. Protein export is required to be a strictly hierarchical process, in which firstly the inner rod and needle filament proteins (early substrates), then needle tip and pore‐forming translocator proteins (intermediate substrates), and finally, after the entire conduit is prepared, effector proteins are secreted and injected (late substrates). Null mutants of the needle length regulator InvJ exhibit a deficiency of inner rod assembly and extremely long needle filaments of uncontrolled length. The loss of the ability to finish needle elongation comes along with a deficiency to switch to the secretion of later substrates in these mutants. Intergenic suppressor mutations of functional null mutants of the flagellar InvJ homolog FliK were found to exclusively locate to the flagellar export apparatus protein FlhB in an early study. FlhB, which corresponds to SpaS in the T3SS of SPI‐1, was later shown to contain a highly conserved N|PTH autocleavage motif. Autocleavage proved to be a prerequisite for switching to the secretion of later substrates but it did not lead to a dissociation of the N‐ and C‐terminal fragments in vitro. We could now show that SpaS autocleavage is critical for substrate specificity switching to occur but that the cleavage event per se does not play a role in this process. Further data suggest that switching utilizes an enhanced flexibility of the C‐terminal domain of SpaS that may lead to its dissociation from the remainder of the protein in vivo. We propose that sufficient flexibility is only achievable by separated polypeptide chains but does not necessarily require an autocleavage mechanism. This hypothesis is corroborated by the finding that invJ suppressor mutations increase the switching propensity of SpaS, possibly by enhancing intradomain flexibility, dispite impaired SpaS autocleavage. 75 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 47 Pathogens and antimicrobial resistance profiles in necrotizing pancreatitis and infected pseudocysts: a retrospective multicenter analysis. H. Albig1, U. Will2, M. Hocke3, T. Bruns1, A. Stallmach1, P. Reuken1 1
Uniklinik Jena, Klinik für Innere Medizin IV, Jena, Germany SRH Waldklinikum Gera, Gera, Germany 3
Helios Klinikum Meiningen, Meiningen, Germany 2
Background Bacterial superinfections in pancreatitis require empiric antibiotic therapy in addition to surgical or endoscopic therapy. Therefore, knowledge of local pathogen pattern and resistance profiles is necessary to choose an optimal antibiotic regimen. Increasing morbidity and frequent endoscopic procedures may lead to an increase in resistant pathogens. Methods Patients suffering from pancreatitis with microbiological conformation of superinfected necrosis or infected pseudocysts in three German hospitals were retrospectively identified. Results 93 patients were included (61 from Gera and 16 each from Jena and Meiningen). Polymicrobial infections was diagnosed in 63 (68%) cases. Most common identified pathogens were Enterococcus spp. in 45 cases including 3 cases with resistance to vancomycin. Streptococcus was identified in 33 cases; E. coli in 15 cases, including 4 infections with of ESBL‐producing pathogens. In 31 (33%) cases Candida albicans was isolated. Most common used antibiotics were cephalosporins (26%), carbapenems (31%) and fluoroquinolones (19%). In vitro resistance of least one of the pathogens towards empiric therapy was detected in 60% of all cases. Reasons for resistant infection were infection with enterococci (38% vs. 19%, p=0.067), whereas infection with E. coli was associated non‐resistant empiric antibiotic therapy (24% vs. 5%, p=0.011). Patients recieving empiric antibiotic therapy with in vitro resistance did not show a higher mortality (p=0.517), but showed a trend to be more often admitted to intensive care unit (50% vs. 35%) and prolonged overall hospital stay (28 vs. 17 days) without reaching statistical significance. Conclusions Empiric antibiotic therapy in patients with infected necrotizing pancreatitis and infected pseudcoysts frequently does not cover isolated pathogen, especialy with enterococci and Candida spp. Ineffective empiric therapy tends to be associated with increased ICU admissions and longer duration of hospitalization. Therefore, empiric therapy should adhere to local antimicrobial resistance as well as individual patients’ risk factors. 76 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 48 Helicobacter pylori induces LTβR signaling in gastric cells R. Mejías‐Luque1, J. Zöller2, F. Anderl1, E. Loew‐Gil2, M. Heikenwälder2, M. Gerhard1 1
2
Technische Universität München, Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Munich, Germany Technische Universität München, Munich, Germany The inflammatory response associated with Helicobacter pylori (H. pylori) infection can act as an initiator of the carcinogenetic process leading to the development of gastric tumors. The cytokines lymphotoxin (LT) α, β and LIGHT belong to the tumor necrosis factor (TNF) superfamily and can activate LTβR mainly expressed on parenchymal and stromal cells. LTβR signaling occurs through the alternative NF‐ĸB signaling pathway, inducing chemokine, cytokine or cell adhesion molecule expression, cell proliferation and cell survival. Although LTβR signaling has been described to support efficient immune responses against pathogens due to maintenance of intact lymphoid structures, no data on LTβR signaling and H. pylori infection has been reported to date. In vitro, the expression of LTs as well as the expression of several chemokines linked to LTβR signaling was induced in human gastric cell lines after H. pylori infection. In line with this, activation of alternative NF‐ĸB pathway was observed. In vivo, the expression of LTα, LTβ and LIGHT was increased and alternative NF‐ĸB activated in the stomach of H. pylori infected humans and mice. Blocking LTβR in mice reduced gastric inflammation. This was accompanied by a higher bacterial colonization of the gastric mucosa. Conversely, agonistic activation of LTβR resulted in an exacerbated inflammatory response. Together our data reveals a novel and crucial role for LTβR signaling executing H. pylori‐induced gastric inflammation. 77 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 49 High failure rate of anti‐Helicobacter pylori triple therapy in Turkish immigrants in Germany M. Sielaff1, G. Demirkaya2, J. Milic2, J. Eick2, J. Steinberg2, W. Ring2, C. Schwertner2, H. Scherübl2 1
2
Vivantes Klinikum Am Urban, Zentrale Notaufnahme, Berlin, Germany Vivantes Klinikum Am Urban, Klinik für Innere Medizin ‐ Gastroenterologie, GI Onkologie und Infektiologie, Berlin, Germany Background Failure of anti‐Helicobacter pylori (HP) therapy is the result of inadequate adherence to therapy and/ or of resistance to the prescribed antibiotics. Methods In a period of seven years (2004‐2011) a total of 144 HP‐positive patients of Turkish descent and with complicated gastroduodenal ulcer disease requiring inpatient treatment in hospital were prescribed triple therapy (pantoprazole 40mg BID, clarithromycin 500 mg BID, amoxicillin 1 g BID for 7 days). In the year 2011 all 144 patients were invited to a 13C‐urea breath test (13C‐UBT). Previous adherence to triple therapy was confirmed by all participating patients. Antimicrobial susceptibility to several drugs (ampicillin, clarithromycin (CLA), metronidazole (MET), tetracycline, levofloxacin (LEV) and rifampicin was tested by the gradient‐diffusion method in gastric biopsies of HP‐positive patients of Turkish descent. Results 63 of the 144 (43 %) patients accepted the invitation and underwent a 13C‐urea breath test. All 63 patients (23 men/ 40 women, mean age 44±15 years in men and 45±16 years in women) confirmed that they had taken HP triple therapy as had been recommended. Nevertheless, 40 of the 63 patients (63,5 %) were found to be HP‐
positive in 13C‐UBT. In the year 2014 microbiologically identified HP strains of 14 consecutive, HP‐positive patients with Turkish descent (second generation) were tested for resistance: 21.4% showed resistance to CLA, 28.6% to MET and 50% to LEV. Conclusion The high rates of both failure of triple therapy and of clarithromycin resistance argue against the empirical use of triple therapy in Turkish migrants. In addition, the high rate of levofloxacin resistance of HP has to be taken into account in Turkish immigrants in Germany, if second line treatment is required. 78 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 50 Dynamic interplay of chronic infections: Concurrent infection of H.pylori and S.mansoni S. Bhattacharjee1, M. Gerhard1, C. Prazeres da Costa1 1
Institute for Medical Microbiology, Immunology and Hygiene, Munich, Germany Helicobacter pylori is estimated to infect about half the world’s population. The pathogen chronically persists in the stomach of individuals and can cause varying disease outcomes ranging from chronic gastritis to gastric and duodenal ulcers as well as gastric cancer. Its strong association with carcinoma has been attributed to the inflammatory response it generates; (Th‐1/Th‐17) via the release of various pro‐inflammatory cytokines. In developing countries where H.pylori infection is ubiquitous, there is also a widespread prevalence of helminths like S.mansoni. The parasite generates distinct immune phases in the host based on its developmental stages and has been shown to alter classical immune responses to other pathogens. Studies have demonstrated that co‐infection with helminths has the potential to skew a pro‐inflammatory response into an anti‐inflammatory one thus delivering protection from conditions such as gastritis. Aim and Results We hypothesized a dependency of the H.pylori induced immune responses on the immune phases generated by S.mansoni and are analyzing the mechanisms enabling this interaction. Furthermore, we also investigated the effect of the bacterial infection on S.mansoni immune responses as well. Focusing on localized (tissue) as well as systemic responses (cytokines) and the interaction between T‐cells and antigen presenting cells in the context of this co‐infection, we aim to understand the bacterial‐helminth crosstalk in what we believe is the niche‐ Peyer’s patches. Our preliminary results show an increased gastric colonization of the bacteria complemented by decreased inflammation in co‐infected mice during the Th‐1 phase of S.mansoni infection. Surprisingly, in the Th2 phase of the helminth infection, H.pylori co‐infection results in decreased granuloma sizes in the liver as well as reduced ALT levels indicative of decreased liver damage. Additionally our in‐vitro assays indicate that the changes in immune responses may be at the level of antigen presentation as well since BMDCs co‐incubated with H.pylori and Schistosome specific SEA show altered cytokine responses. Conclusion and Outlook There is a systemic crosstalk between immune responses generated against H.pylori and S.mansoni with both pathogens influencing the outcome of the other infection. Moreover this immunomodulation is phase dependent. To better decipher the mechanism we will analyze the interaction in the Peyer’s patches in vivo by a ligation system and at the organ level by assessing the microenvironment. Additionally the APC(Min/+) and INS‐GAS mouse models will be used to view how S.mansoni influences the outcome of H.pylori aggravated gastritis. 79 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 51 Neoglycolipids derived from plant oligosaccharides reduce Shiga toxin‐mediated cytotoxicity towards Vero cells G. Pohlentz1, J. Müthing1, P. Dersch2, H. Karch1 1
2
Institut für Hygiene, Universität Münster, Münster, Germany Helmholtz Zentrum für Infektionsforschung, Braunschweig, Germany Enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin (Stx)‐producing E. coli (STEC), give rise to severe and life‐threatening diseases like the hemolytic uremic syndrome (HUS). The initial point for cell destruction by Stxs is their binding to the genuine receptor globotriaosylceramide (Gb3Cer) on the cell surface. The present project aims the development of novel concepts to prohibit Stx from binding to endothelial target cells and thereby to alleviate Stx‐mediated cytotoxicity. Starting from plant polysaccharides a set of oligosaccharides was prepared and transferred to phospholipids to obtain neoglycolipids (neoGLs). Binding of various Stx subtypes to these neoGLs was demonstrated by thin‐layer chromatography overlay assays and individual Stx‐binding neoGLs were identified and structurally characterized by nanoelectrospray ionization mass spectrometry (nanoESI MS). Cytotoxicity assays using Vero‐B4 cells (“gold standard”) and human brain microvascular endothelial cells (HBMECs) were evolved to explore the potential of the plant oligosaccharide‐
derived neoGLs for inhibiting Stx binding to the cell surface receptors and to reduce Stx‐mediated cytotoxicity. NeoGLs were inserted into liposomes and the quality of these supramolecular structures in terms of size and uniformity was checked by dynamic light scattering. Stx1a and Stx2a inhibition assays with Vero‐B4 cells revealed a substantial decrease of Stx‐mediated cellular damage in the presence of neoGL‐containing liposomes. Thus, our newly produced neoGLs form a reliable and promising basis for a novel treatment options for amelioration and prevention of Stx‐mediated diseases. 80 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 52 Leptin regulates macrophage defense against S. Typhimurium infection by regulating Sirtuin1 J. Fischer1,2, S. Gutiérrez3,2, M. Wolke3, R. Ganesan3,2, E. Daglidu3,2, J. Cassens3,2, G. Plum3, P. Hartmann1 J. Brüning4,2, N. Robinson3,2 1
Uniklinik Köln, Klinik 1 für Innere Medizin, Cologne, Germany CECAD, Cologne, Germany 3
Uniklinik Köln, Institut für medizinische Mikrobiologie, Immunologie und Hygiene, Cologne, Germany 4
Max‐Planck‐Institut für Stoffwechselforschung, Cologne, Germany 2
Obese patients exhibit metabolic malfunction and they often suffer from infections indicating a clinically evident dysregulated immune response. Macrophages of the innate immune system are engineered to clear pathogens through phagolysosomal degradation and mount adequate inflammatory response. However, it is poorly understood if metabolic hormones such as leptin play a significant role in tuning the ability of macrophages to attenuate pathogens and to generate appropriate inflammatory response. Sirt1 is a known regulator of metabolic and inflammatory pathways. However, its immunomodulatory role in an altered metabolic condition is unknown. Our investigations revealed that Sirt1 expression is markedly reduced in obese and diabetic patients. Studies in our laboratory have shown that pathogens evade macrophage defense by degrading Sirt1. Interestingly, Sirt1 degradation was stabilized in the leptin‐receptor deficient macrophages upon infection. Furthermore, loss of leptin‐receptor significantly improved the ability of macrophages to kill S. Typhimurium, which correlated with improved phagosome maturation. Also inflammatory response was found to be dampened upon infection with S. Typhimurium as inferred from cytokine estimation and analysis of NFκB and p38 activation. Leptin‐receptor deficiency also prevented S. Typhimurium induced cell death in macrophages. Taken together, we conclude that leptin regulates macrophage defense against pathogens by modulating Sirt1 expression. 81 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 53 Identification of novel adenoviral peptide epitopes and their potential in adoptive T‐cell transfer immunotherapy P. Günther1, J. Peper2, S. Kayser3, B. Faist4, M. Neuenhahn4, D. Busch4, T. Feuchtinger5, S. Stevanović2 K. Dennehy1 1
Universitätsklinik Tübingen, Med Virologie, Tübingen, Germany University of Tübingen, Department of Immunology, Tübingen, Germany 3
University Children’s Hospital Tübingen, Pediatric Hematology/Oncology, Tübingen, Germany 4
Technical University Munich, Institute for Medical Microbiology, Immunology and Hygiene, Munich, Germany 5
Ludwig Maximilian University, Hematology and Oncology, Dr. von Haunersches Children’s Hospital, Munich, Germany 2
Adenovirus infections of immuno‐compromised patients, particularly following allogeneic haematopoietic stem cell transplantation, are associated with morbidity and mortality. Immunotherapy by adoptive transfer of hexon‐ and penton‐specific T‐cells has been successfully applied, but many approaches are impeded by the low number of HLA class I‐restricted adenoviral peptide epitopes described to date. We use a novel method to identify naturally presented adenoviral peptide epitopes from infected human cells, ectopically expressing defined HLA, using peptide elution and liquid chromatography‐mass spectrometry analysis. We show that the previously described HLA‐A*01:01‐restricted peptide epitope LTDLGQNLLY from hexon protein is naturally presented, and demonstrate the functionality of LTDLGQNLLY‐specific T‐cells. We further identify a novel immunodominant HLA‐B*07:02‐restricted peptide epitope VPATGRTLVL from protein 13.6K, and demonstrate the high proliferative, cytotoxic and IFN‐g producing capacity of peptide‐specific T‐cells. Lastly, LTDLGQNLLY‐
specific T‐cells can be detected ex vivo following adoptive transfer therapy, and LTDLGQNLLY‐ and VPATGRTLVL‐specific T‐cells have memory phenotypes ex vivo. Given their proliferative and cytotoxic capacity, such epitope specific T‐cells are promising candidates for adoptive T‐cell transfer therapy of adenovirus infection. 82 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 54 Disseminated Mycobacterium Avium Complex Infection manifesting with excessive weight loss and abdominal pain in an AIDS Patient: a case report C. Malainou1, T. Discher1, J. Lohmeyer1, S. Gattenlöhner2 1
2
Universitätsklinikum Giessen und Marburg, Infektiologie, Giessen, Germany Universitätsklinikum Giessen und Marburg, Pathologie, Giessen, Germany Background Nontuberculous mycobacterial infections though rare in immune competent individuals can be a serious cause of morbidity for immune deficient patients. They usually follow a disseminated and rapidly deteriorating course and should thus be diagnosed early by internists treating AIDS patients. Case Presentation This is the case of a 35‐ year old Caucasian male patient with a history of noncompliance regarding the combined antiretroviral therapy. The patient presented himself in our Outpatient Clinic with severe abdominal pain, fever, weight loss and fatigue. The laboratory results revealed a desolated immune status, microcytic anemia as well as increased serum transaminase and alkaline phosphatase levels. Diagnosis was confirmed after performing liver biopsy, whose histopathological and microbiological findings were consistent with a Mycobacterium avium complex infection. The patient was subsequently treated with ethambutol, clarithromycin and rifabutin, whereas the early start of the antiretroviral therapy was the key factor to his successful recovery. Conclusion Poor compliance in patients diagnosed with the human immunodeficiency virus is a common phenomenon with potential detrimental consequences. Patient commitment should thus be continuously reevaluated and syndrome related illnesses should be early detected. An invasive diagnostic approach can sometimes be the key to a successful treatment. Figure 1 Figure 2 83 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 55 The DZIF Transplantation Cohort e.V. D. Schindler1 1
Klinikum rechts der Isar, Munich, Germany Infections in transplant recipients have a decisive impact on graft function and survival of the transplant recipient, but many issues are still poorly understood. For example, little is known about the long‐term consequences of many infections on graft survival/function and graft‐ versus‐host disease (GvHD), the role of individual susceptibility to bacterial, viral and fungal colonisation under immune suppression, the evolution of the antimicrobial T cell repertoire, the long‐ term impact of anti‐infective therapy on graft and patient survival or changes in the physiological microbiome that may be significant for colonisation with pathogenic and antibiotic‐resistant microbes. Taking advantage of the fact that the university hospitals which are partners in DZIF have sizeable transplantation programs, we establish a large multicentre prospective observational cohort of transplant recipients. A prospective cohort study would allow correlating infection with and the immune response to the development of transplant complications in a prospective manner. Worldwide, there are very few prospective cohort studies on transplant patients with a focus on infectious disease using a standardized protocol for the collection of many different samples at defined time points before and after transplantation. To allow careful epidemiological analysis of the impact of infections on transplant function and survival, a large prospective cohort based on systematic criteria for enrolment, data acquisition is needed. In addition to clinical data, a range of biological samples will be collected and stored on a regular basis. The cohort study is designed such that it will be open to using modern genomic and epigenomic technology. Following extensive discussions among the participating DZIF institutions, a study protocol has been agreed, consent from the local institutional review boards and data protection officers have been obtained, a databank for collecting patient data and information on stored biosamples has been programmed. The DZIF transplant cohort has been registered as an association (‘eingetragener Verein’) and the relevant committees (executive board, scientific steering committee, general assembly) have been established and have met on several occasions. The first pilot study has already taken place at all participating centers and is currently being extended. 84 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 56 1,3‐ß‐D‐glucan in infusion solutions B. Liss1, H. Wisplinghoff2, D. Hoffmann3, V. Dimitriou4, O. A. Cornely1 1
Uniklinik Köln, Klinik I für Innere Medizin, Hämatologie und Onkologie; Studienzentrum Infektiologie II, Cologne, Germany Uniklinik Köln, Institut für Mikrobiologie, Cologne, Germany 3
Wisplinghoff Laboratories, Cologne, Germany 4
German Centre for Infection Research (DZIF), Cologne, Germany 2
Background 1,3‐ß‐D‐glucan (BDG) is a fungal cell wall constituent and is an acknowledge microbiological criterion used to diagnose invasive fungal infections (IFI). The optimal cut‐off to positivity remains elusive. Certain blood products and anti‐neoplastic drugs are associated with false‐positive tests. Methods To evaluate the potential for interaction the most frequently applied infusion types were selected. A total of four blood products, 37 anti‐infectives and 5 anti‐neoplastic drugs were tested for their BDG content using two methodologically different BDG assays (Fungitell Assay, FA (Associates of Cape Cod, MA, USA) and BioAssay ELISA Kit (BA, United States Biological, Salem, MA, USA)). In addition, expected post transfusion serum levels were calculated and compared to actual serum BDG levels pre and post transfusion in patients without IFI, who had signed informed consent to biobanking (Cologne No. 33‐08). Results BDG was found in 3 of 4 blood products, 26 of 37 (70.0%) anti‐infectives, including all antifungals, and 5 of 5 antineoplastic drugs. Concentrations were sufficiently high to cause a positive BDG test after infusion of a standard dose of: Fresh frozen plasma, human albumin, packed red cells and pegylated asparaginase. We calculated expected post‐infusion levels for these drugs and assessed serum BDG concentration pre and post infusion (table 1). For pegylated asparaginase, the drug with the highest BDG concentration, we drew serial blood samples every 24h to determine kinetics (figure 1). Conclusion Many common blood products and drugs are BDG contaminated. BDG concentrations measured in infusion solutions can be used to approximately predict the magnitude of BDG serum levels increase in patients. Adjusting the formula to accommodate the patient’s weight and height might yield improved predictions. BDG levels remain elevated for several days after infusion of a contaminated solution. Individual correction of the BDG cut‐off to positivity for each patient based on the received infusions and blood products could improve specificity without lowering sensitivity. Figure 1 Figure 2 85 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 57 Inhibitor development of the interaction between the LMP1 oncoprotein of Epstein‐Barr virus and TRAF proteins F. Giehler1,2, K. Schorpp3, K. Kopp4, S. Dornauer3, I. Antes4, K. Hadian3, A. Kieser1,2 1
DZIF, Partner Site Munich, Munich, Germany Helmholtz Zentrum Munich, Research Unit Gene Vectors, Munich, Germany 3
Helmholtz Zentrum Munich, Assay Development and Screening Platform, Neuherberg, Germany 4
Technische Universität Munich, Theoretical Chemical Biology and Protein Modelling Group, Freising, Germany 2
Lymphomas associated with Epstein‐Barr virus (EBV) infection, such as post‐transplant lymphoproliferative disease (PTLD), are severe complications in immunocompromised patients. No drug is available that targets EBV. For cell transformation, the virus relies on the expression of its oncogene latent membrane protein 1 (LMP1). The intracellular C‐terminus of LMP1 harbors two C‐terminal activator regions, CTAR1 and 2, which are both essential for cell transformation by the virus and interact with TNF‐receptor associated factor (TRAF) proteins to activate cellular signaling pathways including NF‐kappaB. CTAR1 directly recruits TRAF2, whereas CTAR2 requires TRAF6 for signaling. Here we aim at the development of small molecule inhibitors for the interaction of LMP1 with critical TRAF proteins to disonnect LMP1 from its transforming signaling network and thus counteract EBV‐associated disease. We developed high throughput screening (HTS) compatible assay technology based on the AlphaScreen detection system and the protein components GST‐LMP1 and His‐TRAF2. This very stable mix‐and‐measure assay has a Z' of 0.6 > Z' > 0.9 in 384‐well format. In a pilot screening performed at the HMGU we identified a new compound, dubbed EN06, which inhibited LMP1::TRAF2 interaction and interfered with proliferation of EBV‐transformed human B‐cells. This result delivered proof of principle that LMP1::TRAF interactions are suitable drug targets. EN06 derivatization revealed that a carboxyl group of EN06 is essential for interaction with the target TRAF2. Target and assay have recently been accepted at the European Lead Factory (ELF), a private public partnership of the EU and the European Federation of Pharmaceutical Industries (EFPIA), for ultra HTS of 350,000 compounds derived from industrial libraries. The assay has been downsized and transfered successfully to the ELF screening center. The primary screening campaign and the subsequent generation of a qualified hit list is ongoing. To block CTAR2 by small molecules, we sought to identify a targetable protein‐protein interaction (PPI), which is critical for activity of this domain. Currently it is believed that LMP1 recruits TRAF6 by an indirect mechanism. We purified recombinant TRAFs 1‐6 to characterize their interaction with LMP1 in cell‐free systems. Surprisingly, TRAF6 bound to CTAR2 via a direct protein‐protein interaction. We characterized this interaction in detail by mutational analysis and dynamic structural modeling. Functional relevance of this interaction for LMP1 activity was demonstrated by rescue experiments with TRAF6 wildtype and interaction‐defective mutants in TRAF6‐deficient cells. Based on the protein components GST‐LMP1 and His‐TRAF6 we established a first‐generation PPI assay based on the AlphaScreen technology, which will be further developed to uHTS‐
compatible format. 86 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 58 High quality biosample collection within the German Center for Infection Research: the Transplantation cohort as pilot project for IT, ELSI and QM questions A. Kuehn1,2, G. Anton1,2, L. Glück3,4, D. Schindler2,5, D. Busch2,5, P. Schirmacher3,4, H.‐ E. Wichmann1,2 C. Gieger1,2 1
Helmholtz Zentrum Munich, EPI2/AME, Neuherberg, Germany Deutsches Zentrum für Infektionsforschung (DZIF), Standort München, Munich, Germany 3
Universitätsklinik Heidelberg, Institut für Pathologie, Heidelberg, Germany 4
Deutsches Zentrum für Infektionsforschung (DZIF), Standort Heidelberg, Heidelberg, Germany 5
Universitaetsklinikum Rechts der Isar, Munich, Germany 2
The availability of quality‐controlled, standardized, and well‐documented biomaterials is a precondition for the collaborative translational research at the German Center for Infection Research (DZIF). The Infrastructural Unit (TI) Biobanking has been established as a central core function of DZIF to provide rapid and reliable access to human tissue and liquid samples. Its central project will be the support of the Transplantation (Tx)‐cohort within the TTU Infections of the immunocompromised Host (IICH). Beginning in 2015 with a pilot phase, the routine cohort activities will start in 2016. The recruitment takes place at four DZIF sites, the organs kidney, liver, heart, stem cells, pancreas and lung are included. Relevant medical and associated data will be stored in a self‐tailored database; biosample‐associated data will be stored locally in the individual Laboratory Management Information Systems (LIMS) of the partner sites. The focus of the TI Biobanking in this context will be on data management by implementing a central LIMS at the Munich site. This central LIMS will function as a comprehensive catalogue extracting a defined data set (metadata and preanalytical data) from the local LIMS. Thus, it can be used as search engine for all DZIF biosamples. Concerning quality management, common and harmonized standard operating procedures (SOPs) and/or minimal standards for sample retrieval, sample processing, transport and storage of biosamples taking into account the demands of the clinical context will be developed. Additionally, the training of local staff for internal audits and audit coordination will be provided, as well as support in ELSI questions. 87 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 59 Characterization of HLA‐C*07‐restricted Cytomegalovirus‐specific CD8+ T cells F. Schlott 1, D. Steubl2, S. Ameres 3, A. Mossmann3, S. Dreher1, U. Heemann2, D. Busch1, M. Neuenhahn1 1
med. Mikrobiologie, Immunologie und Hygiene, TUM, Munich, Germany Klinikum rechts der Isar, TUM, Department of Nephrology, Munich, Germany 3
Helmholtz Zentrum Munich, Clinical Cooperation Group Immunooncology, Munich, Germany 2
Human Cytomegalovirus (CMV) infection remains a major source of morbidity and mortality in patients after solid organ‐ or hematopoietic stem cell transplantation (HSCT). Control of viral replication depends on cytotoxic T cells, which recognize CMV epitopes in the context of an individual HLA‐repertoire. Adoptive T cell therapy with CMV‐specific T cells is currently evaluated for HSCT recipients, but might be counteracted by CMV’s immune evasion strategies. HLA‐C*07:02‐CMV epitope‐restricted CD8+ T cells are an interesting target for adoptive T cell therapy, as this HLA‐molecule is less susceptible to viral immune evasion. It is very common in the Caucasian population (ca. 30%), where HLA‐C*07:02 is nearly always coexpressed with HLA‐B*07:02. To better understand this promising T cell population, we used the HLA‐C*07:02‐restricted IE‐1309‐317 epitope for the generation of reversible multimers (C7/IE‐1‐Streptamers). We analyzed the phenotype and functional potential of C7/IE‐1‐Streptamer+ T cells and compared the findings with Streptamer‐specific T cells to the well‐
known HLA‐B*07:02‐restricted pp65417‐426 epitope (B7/pp65‐Streptamers). Using both Streptamers in a cohort of 20 healthy individuals we found higher frequencies of C7/IE‐1 (mean = 14.9%) in comparison to B7/pp65‐restricted T cells (mean = 2.7%; p < 0.001), which was also confirmed by IFNγ secretion in intracellular cytokine staining (C7/IE‐1, mean = 6.7%; B7/pp65, mean = 1.8%; p = 0.01). However, correlation for multimer‐positive and IFNγ‐secreting T cells was intermediate for C7/IE‐1 (r2 = 0.77) but high for B7/pp65 (r2 = 0.90). We analyzed Killer immunoglobulin‐like receptor (KIR) expression on CD8+ T cells and found epitope‐independent KIR2DL2/3 binding of C7/IE‐1‐Streptamers. We therefore generated HLA‐C7 multimers refolded with an irrelevant peptide (C7/MAGE‐Streptamer), and performed double staining to exclude KIR‐expressing CD8+ T cells. After reanalyzing the samples we found lower numbers of single C7/IE‐1‐
Streptamer+ T cells (9.97%), which now strongly correlated (r2 = 0.96) with IFNγ‐secreting T cells. In addition, we analyzed the phenotype of HLA‐C‐restricted T cells. C7/IE‐1‐restricted CD8+ T cells contained substantial numbers with a central memory phenotype qualifying them for expansion after adoptive T cell transfer. Taken together, we successfully generated C7/IE‐1‐Streptamers and identified C7/IE‐1‐restricted T cells as a large and functional T cell population. They contained both central and effector memory phenotypes. HLA‐
C*07:02/IE‐1‐restricted T cells are in consequence a potent candidate for adoptive T cell therapy of CMV‐
infections, but misbinding to KIR2DL2/3‐expressing CD8+ T cells needs to be controlled (e.g. by serial MHC Streptamer purification). 88 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 60 Primary Cytomegalovirus infection in seronegative kidney transplant patients is associated with protracted cold ischemia time of seropositive donor organs F. Schlott 1, D. Steubl 2, J. Albrecht1, E. Matevossian3, J. Lutz4, U. Heemann2, L. Renders2, D. Busch1, M. Neuenhahn1 1
Technische Universität München, Institute for Medical Microbiology, Immunology and Hygiene, Munich, Germany Technische Universität München, Department of Nephrology, Klinikum rechts der Isar, Munich, Germany 3
Technische University München, Department of Surgery, Klinikum rechts der Isar, Munich, Germany 4
Johannes‐Gutenberg University Mainz, Section of Nephrology, I. Department of Medicine, Mainz, Germany 2
Human Cytomegalovirus (CMV) infection can cause serious complications in immunocompromised patients. In kidney transplant recipients, CMV infection or reactivation can lead to organ rejection, secondary infections or single/multi‐organ failure. Depending on the CMV IgG serostatus patients are divided into several risk groups with donor+/recipient‐ (D+/R‐) carrying the highest risk for CMV‐replication. However, risk factors predisposing for primary infection of CMV‐seronegative NTx recipients are still not fully elucidated. In order to investigate this issue, we prospectively monitored 8 high‐risk NTx patients for the incidence of acute CMV viremia using PCR and measured CMV‐specific immunity by QuantiFERON assay and intracellular cytokine staining. In addition, we analyzed the CMV‐specific antibody response and infection status by CMV IgG/IgM‐specific ELISA. 4 out of 8 patients developed relevant CMV‐replication (>500 copies/ml) leading to CMV‐associated disease in three of these patients. Replication occurred after termination of a 3 month antiviral prophylaxis with Valganciclovir in all of these patients. In addition, we detected induction of CMV‐specific T cell immunity against immunodominant epitopes (pp65 and IE‐1) in 3 of 4 patients and all patients with CMV‐replication showed IgG or IgM seroconversion. In contrast, patients without viremia neither established CMV‐specific T cells nor CMV‐specific antibodies and remained therefore most likely uninfected despite reception of a CMV‐
seropositive transplant. When we compared the cold and warm ischemia times of both groups we found significant differences in the cold ischemia time for patients with (mean = 960 min; 720‐1080 min) and without (mean = 362.5 min; 120‐660 min; p = 0.0286) CMV‐replication. In contrast, warm ischemia time did not differ significantly between both groups (mean = 31.5 min; 16‐60 min vs. mean = 21.5 min; 16‐30 min p = 0.6532). Taken together, our results show that cold ischemia time may predispose for acute CMV‐infection in D+R‐ NTx patients. In consequence, high risk patients with protracted cold ischemia time should be thoroughly monitored for CMV‐replication well beyond completion of antiviral prophylaxis. 89 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 61 Diagnostic Metagenomics from BAL Samples significantly improves the Management of Allogenic HSCT Patients with Pulmonary Complications A. Grundhoff1, H. Lellek2, E. M. Klupp3, M. Alawi1, D. Indenbirken1, M. Lütgehetmann3, M. Christner3, N. Kröger2 H. Rohde3, N. Fischer3 1
Heinrich‐Pette Institut, Leibniz Institut für experimentelle Virologie, Virus Genomik, Hamburg, Germany Universitätsklinikum Hamburg, Klinik für Stammzelltransplantation, Hamburg, Germany 3
Universitätsklinikum Hamburg‐Eppendorf, Inst. f. Med. Mikrobiologie, Virologie und Hygiene, Hamburg, Germany 2
Despite of improved prophylaxis and conventional microbiological diagnostics pneumonia remains a significant cause of mortality after hematopoietic stem cell transplantation (HSCT): detection of an infectious agent fails in more than 50% of cases. Unbiased, non‐targeted metagenomic RNA sequencing (UMERS) represents an unbiased method which can detect known, but also distantly related or even novel pathogens of viral, bacterial, fungal or parasitic origin. We here apply UMERS retrospectively to 24 respiratory specimens of 21 HSCT patients with CT confirmed pulmonary lesions. UMERS was systematically compared to conventional diagnostic tests including bacterial/fungal culture and multiplex PCR. In contrast to conventional diagnostics (pathogen detection rate 33.3%), UMERS detected in 58.3% of the samples pathogens with the potential to cause respiratory diseases. All pathogens detected by conventional tests were also faithfully identified by UMERS. Additionally, UMERS revealed one viral infection with human parainfluenza 3, four samples showed clear signs of bacterial infection (high abundance of bacterial reads) and three samples contained fungal sequences, Scedosporium apiospermum, Fusarium sp., both can cause fatal pulmonary complications in immunosuppressed patients. UMERS significantly contributes to the improvement of diagnostics and support of epidemiological analysis of putative nosocomial infections and thus enhances the level of recognition and treatment of pulmonary complications. 90 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 62 Modified Vaccinia virus Ankara delivered HCMV antigens stimulate proliferation and activation of HCMV‐specific CD8+ T cells E. Schober1,2, M. Lehmann1,2, S. Ameres2,3, C. Brandmüller1, A. Moosmann2,3, G. Sutter1,2 1
Ludwig‐Maximilians‐University Munich , Institute for Infectious Diseases and Zoonoses, Munich, Germany German Centre for Infection Research, Munich, Germany 3
Helmholtz Zentrum Munich, Clinical Cooperation Group Immunooncology, Munich, Germany 2
Infection with human cytomegalovirus (HCMV) affects a majority of humans worldwide. HCMV is efficiently controlled by the immune system of a healthy individual, but can be severely pathogenic in immunocompromised patients, for example after transplantation. The HCMV proteins pp65 and immediate‐
early‐1 (IE‐1) are among the most important targets of the HCMV‐specific CD8 and CD4 T cell response. The orthopoxvirus strain Modified Vaccinia virus Ankara (MVA) does not replicate in most mammalian cells, exerts strong adjuvant properties, and foreign genes can be introduced into its genome, which are efficiently expressed in vitro and in vivo. We generated a recombinant MVA expressing a pp65‐IE‐1 fusion gene for use as a candidate HCMV vaccine. The nuclear localization signal and the STAT3 binding region of HCMV IE‐1 were deleted to prevent signalling activity while maintaining major T cell epitopes. We found that MVA‐HCMV_pp65‐IE‐1 is able to mediate presentation of HCMV epitopes to CD8+ T cells. Using MVA‐infected autologous B cells, we reactivated HCMV‐specific T cells from human peripheral blood. MHC‐
peptide multimer staining showed that CD8+ T cells specific for various epitopes from pp65 and IE‐1 were efficiently co‐expanded after only one round of stimulation, achieving an up to 100‐fold specific CD8+ T cell expansion compared to controls. For each of the two antigens, MVA‐HCMV_pp65‐IE‐1 induced the same level of specific CD8+ T cells as viruses encoding a single HCMV antigen, MVA‐pp65 and MVA‐IE‐1. Our results indicate that MVA is an effective vector for delivering HCMV antigens to antigen‐presenting cells for induction of HCMV‐specific cytotoxic T cell responses. 91 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 63 Evidence for presence of latent human cytomegalovirus in microvascular endothelial cells of human lung and liver tissue B. Kasmapour Seighalani1, Z. Chaudhry1, B. Wiegmann2, A. Haverich2, F. W. R. Vondran3, F. Klann1, L. Cicin‐Sain1 1
Helmholtz‐Zentrum für Infektionsforschung , IMCI, Braunschweig, Germany Hannover Medical School, Department for Cardiothoracic, Transplantation and Vascular Surgery, Hannover, Germany 3
Hannover Medical School, Abdominal and Transplant Surgery, Hannover, Germany 2
Human cytomegalovirus (HCMV) infection is ubiquitous, with up to two thirds of population latently infected. CMV reactivation could occur in vulnerable cohorts such as immunocompromised and solid organ transplantation recipients, leading to serious and long‐term complications, however, all reservoir cells for the latent virus, as well as molecular mechanisms ushering the CMV reactivation are not fully characterized. We have isolated, cultivated and preserved a combined total of 50 distinct lung microvasculature endothelial (HMVEC‐L) and liver sinusoidal endothelial (LSEC) cells, from lung transplant recipients and patients undergoing partial liver resections receptively, as potential repositories of latent CMV. Using nested PCR for ie1 gene of HCMV, viral DNA was detected in virtually all seropositive samples, suggesting presence of latent CMV genome in the lung and liver endothelial cells. Viral genome in cells isolated from various donors followed different dynamics, but remained detectable in endothelial fraction even after long cultivations of up to two months despite the inevitable dilution of the cells due to repeated passaging. This suggests activation and replication of the virus. Preliminary RT‐PCR assays confirm this and show ie1 mRNA is present in endothelial cells of CMV seropositive patients after 21‐28 days of cultivation. However, an overt CPE effect is not observed in the endothelial cells, suggesting possibly the adaptation of the virus to its host cells or a very slow virus spread process. In the following months, we will explore the dynamics of CMV infection in the isolated primary endothelial to; a) confirm the observed infection is a bona fide reactivation of latent CMV in endothelial cells, b) characterize the time‐line, viral load and viral activity of such a reactivation, c) establish an in vitro model of latency in endothelial cells and d) what factors contribute to promoting and inhibiting virus reactivation. 92 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 64 FungiScopeTM ‐ Global Emerging Fungal Infection Registry D. Seidel1,2, L. A. Durán Graeff2, M. J. G. Vehreschild1,2, B. Liss1,2, P. Köhler1,2, F. Müller1,2, H. Wisplinghoff3 J. J. Vehreschild1,2, O. A. Cornely1,4,5,2 1
German Center for Infection Research, Partner Site Bonn‐Cologne, Department I of Internal Medicine, Cologne, Germany University of Cologne, Center for Integrated Oncology CIO Cologne/Bonn, Cologne, Germany 3
University of Cologne, Institute for Medical Microbiology, Immunology and Hygiene, Cologne, Germany 4
University of Cologne, Clinical Trials Centre Cologne, ZKS Cologne, BMBF 01KN1106, Cologne, Germany 5
University of Cologne, Cologne Excellence Cluster on Cellular Stress Responses in Aging‐Associated Diseases (CECAD), Cologne, Germany 2
Background Number of rare invasive fungal infection (IFI) are rising worldwide due to increasing patient population at risk. To broaden knowledge on epidemiology of emerging IFD, FungiScope a global registry has been initiated. Currently, partners from 60 countries contribute cases that eventually help determining clinical patterns, improve diagnostic procedures and therapeutic regimens. Methods FungiScope uses web‐based data capture accessible through www.fungiscope.net. For case enrollment, cultural, histological, antigen or molecular evidence on the occurrence of infection with non‐endemic fungi is required. Data collected include demographics, underlying conditions, immunosuppressive medication, clinical signs and symptoms, sites of infection, diagnostic tests, pathogen identification, antifungal treatment and outcome. Clinical isolates are collected for centralized identification, molecular analyses and exchange between collaborators. Results To date, 429 cases have been captured. Mucorales (n=196; 45.7%), Fusarium spp. (n=65; 15.2%), yeasts (n=54; 12.6%), and dematiaceae (n=48; 11.2%) are the most frequently registered pathogens. Chemotherapy (n=195; 45.5%) and stem cell transplantation for hematological malignancy (n=102; 23.8%) were the predominant risk factors, followed by intensive care (n=95; 22.1%), diabetes mellitus (n=84; 19.6%), and chronic renal disease (n=35; 8.2%). For 20 cases (4.7%) no risk factor was identified. Major sites of infection included lung (n=214; 49.9%), paranasal sinuses (n=79; 18.4%), blood stream (n=77; 17.9%), and deep soft tissue (n=65; 15.2%). Disseminated infection (n=72, 16.8%) was mostly associated with lung (n= 51, 70.8%), blood stream (n= 31, 43.1%) and CNS involvement (n= 24, 33.3%). For 212 (52.6%) patients, complete or partial response to treatment of IFD was documented. All‐cause‐mortality and mortality attributable to IFD was 45.2% and 34%, respectively. Conclusion The clinical relevance of emerging IFD is increasing. FungiScope is a vividly expanding network that attained increasing interest throughout the years. In a short time period, a wide variety of cases has been collected that provide a comprehensive view on the epidemiology and clinical presentation of rare IFI. Keywords Rare fungal infections, web‐based register, epidemiology 93 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 65 Posaconazole does not affect on functional capacities of human polymorphonuclear neutrophils and human monocyte derived macrophages in vitro F. Farowski1, O. A. Cornely1,2,3,4,5, P. Hartmann1,5,6 1
Uniklinik Köln, Klinik I für Innere Medizin, Cologne, Germany Universität zu Köln, Zentrum für Klinische Studien (ZKS Cologne, BMBF 01KN0706), Cologne, Germany 3
Uniklinik Köln, Centrum für Integrierte Onkolgie (CIO), Cologne, Germany 4
Universität zu Köln, Cologne Excellence Cluster on Cellular Stress Responses in Aging‐Associated Diseases (CECAD), Cologne, Germany 5
Deutsches Zentrum für Infektionsforschung (DZIF), Cologne, Germany 6
Uniklinik Köln, Institut für Medizinische Mikrobiologie und Hygiene (IMMIH), Cologne, Germany 2
We previously demonstrated that the intracellular concentration of posaconazole in peripheral blood mononuclear cells and polymorphonuclear neutrophils (PMN) is significantly increased (22.5 and 7.66 fold, respectively) compared to the plasma concentration. Given the importance of these cells in the immune response to fungal infections we were wondering whether the intracellular accumulation of posaconazole impacts on their functional capacities. Various studies proposed immunomodulatory properties for other azole antifungal drugs like fluconazole and itrconazole. Therefore, we systematically investigated if and how posaconazole impacts on the functional capacities of PMNs and human monocyte derived macrophages (MDM). In particular, we measured migration of posaconazole loaded PMNs (chemotaxis in vitro), the release of reactive oxygen species (ROS) from and the phagocytic capacities, as well as the killing capacities, of PMNs and MDMs in the presence of posaconazole (0.2, 0.6, and 1.2 µg/mL). Despite high intracellular concentrations of Posaconazole PMNs and MDMs performed equally well regarding all functional capacities when compared to naïve control cells. We only observed a reduced release of ROS by neutrophils after stimulation with of A. fumigatus conidia for three hours. We attributed this effect rather to reduced metabolic activity, i.e. germination of the A. fumigatus conidia in response to the drug than to a immunmodulatory effect of posaconazole. In fact, when stimulated with E. coli, neutrophils showed equal ROS release in the presence and absence of posaconazole. However, the observation of decreased ROS release in the presence of posaconazole may indicate that patients who experience control of fungal germination by sufficient drug levels suffer less collateral tissue damage at the side of infection associated with excessive ROS release. 94 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 66 Genetic analysis of a patient cohort with chronic mucocutaneous candidiasis N. Frede1, M. Depner1, J. Rojas1, K. Hübscher1, B. Grimbacher1 1
Universitätsklinik Freiburg, Centrum für chronische Immundefizienz, Freiburg, Germany Chronic mucocutaneous candidiasis (CMC) constitutes a primary immunodeficiency characterized by an enhanced susceptibility to infections caused by candida ssp. CMC is associated with a significant morbidity due to recurrent or persistent skin, nail and mucous membrane involvement. While disseminated invasive infections are rare, they constitute a dreaded complication due to the high associated mortality rate. During recent years, a growing number of mutations underlying the clinical phenotype of CMC have been identified, which highlight the importance of the Th17 axis (STAT1, ACT1, IL‐17F, IL‐17RA) as well as pattern recognition receptor signalling (Dectin‐1/CARD9) in antifungal immunity. Due to the clinical heterogeneity of CMC the establishment of a genetic diagnosis is essential for determining the prognosis and ideal treatment for individual patients. Therefore, we have established targeted panel re‐
sequencing in order to facilitate the time‐ and cost‐effective identification of underlying genetic defects. Specifically, we have designed a gene panel comprising 43 genes associated with CMC, Hyper‐IgE syndrome and chronic granulomatous disease, i.e. three primary immunodeficiencies with overlapping clinical phenotypes including the susceptibility to fungal infections. Target enrichment was performed with the help of Agilent’s HaloPlex technology followed by sequencing on Illumina’s MiSeq system. All detected mutations were confirmed by Sanger sequencing. To date, we have collected a cohort of 24 kindreds with familial CMC including at least 57 cases of verified CMC as well as 46 sporadic patients. Out of the 24 kindreds 13 were shown to have STAT1 mutations, four families had mutations in CARD9, while one family had a mutation in IL17RA. Six families did not show a mutation in any of the known candidate genes. Out of these, three families underwent whole exome sequencing as an unbiased approach in order to identify potential novel genetic defects. Evaluation of novel candidate genes is ongoing. Out of the 46 sporadic CMC patients 14 were shown to have STAT1 mutations, while two patients had CARD9 mutations. Two other patients were demonstrated to have STAT3 mutations and one patient had a compound heterozygous CARD9 defect as well as a STAT1 mutation. For ten patients the results of targeted panel re‐
sequencing are pending. The analysis of larger patient cohorts will allow for a more precise definition of the different disease entities subsumed under the diagnosis of CMC. Furthermore, the molecular characterization of these genetic defects constitutes a prerequisite for the development of targeted drug therapy as well as novel therapeutic approaches such as gene surgery. 95 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 67 HHV‐6‐specific CD8+ T cells target a wide range of antigens L. Martin1, A. Moosmann1 1
Helmholtz Zentrum Munich, Munich, Germany Human herpesvirus 6 is very widespread in the human population, causes significant morbidity in immunocompromised patients, and is likely involved in autoimmune disease. As for other herpesviruses, a prominent role of specific cytotoxic CD8 T cells in antiviral control is assumed, but knowledge about the HHV‐6‐
specific CD8 T cell repertoire has been limited. Thus, we undertook a cross‐sectional analysis of CD8 T cell responses to HHV‐6B, the most widespread HHV‐6 species. To identify possible target antigens of this virus, we performed a screen with all possible 299 peptides that contain a typical HLA‐B*08:01 binding motif. Such epitope candidates were present in 79 of 98 unique viral proteins or ORFs. We used these peptides to expand CD8 T cells from healthy carriers, and established CD8 T cell clones. A large majority of T cell clones recognized HHV‐6B‐infected cells. Thirteen epitopes from 10 proteins from all phases of the viral replication cycle were presented by infected cells to CD8 T cells, including the immediate‐early 1 protein, the DNA polymerase, the major DNA‐binding protein, and proteins of unknown function or hypothetical expression. Moreover, seven of seven tested epitopes were presented by HHV‐6A‐
infected cells. While the cloning approach showed that individual donors harbored more than ten epitope specificities, ex vivo detection by HLA‐peptide multimers identified a median of six specificities per donor, all at frequencies below 0.1% of CD8 T cells. Thus, a herpesvirus‐specific T cell repertoire can be efficiently characterized by a proteome‐wide peptide‐based screen. Although considering only one HLA restriction, we find that HHV‐6B‐specific CD8 T cell repertoires are composed of many specificities targeting epitopes from various classes of proteins, in the absence of any prominent immunodominance focused on certain epitopes or functional classes of proteins. Thus, CD8 T cell responses to HHV‐6B and other herpesviruses are differentially structured. The implications of these findings for adoptive immunotherapy will be discussed. 96 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 68 Novel neutralizing antibody with clinical potential against Epstein‐Barr‐virus infections in the immunocompromised host. K. Gärtner1, M. Kellner1, R. Feederle1, J. Dünzkofer2, E. Kremmer1, R. Stripecke3, R. Zeidler2 1
Helmholtz‐Zentrum Munich, Munich, Germany Klinikum der Universität, Munich, Germany 3
Medizinische Hochschule, Hannover, Germany 2
EBV is a widespread human herpesvirus that is mostly harmless but regularly leads to morbidity and mortality in patients immunocompromised due to immune defects or transplantations, where it is associated with lymphoproliferative disorders. Antibody‐based immunotherapy is a promising approach for an immediate adoptive protection against viral infections and development of associated diseases. Consequently, passive immunization with virus‐specific neutralizing antibodies that block infection and/or interfere with virus spreading has been regularly explored for prophylaxis of infection‐associated diseases in patients with inherited, acquired or iatrogenic immune defects. Exosomes are membrane‐enclosed, highly immunogenic vesicles that are released by many types of cells. Using our proprietary immunization technology based on the use of engineered exosomes, we generated a new monoclonal antibody (calles 6G4) targeting the EBV glycoprotein gp350. For a clinical application at a later time point, we have successfully cloned the variable regions of the immunoglobulin chains encoding for the heavy (IgH) and light (IgL) chain of this antibody and fused them to a human IgG1 Fc‐fragment, yielding a chimeric IgG1 antibody. In vitro, our new gp350‐specific monoclonal antibody (mAb) neutralizes EBV and protects human B cells from infection much more efficiently than established gp350‐antibodies. Because 72A1, an established gp350 antibody, can prevent development of EBV‐positive tumors in humanized mice and provide partial short‐term protection against EBV in seronegative liver transplant recipients, we will test the potential of our new antibody to block infection and to protect from EBV‐associated disease in a relevant humanized animal model. Neutralizing mAbs active against viral pathogens, including EBV, are of great clinical interest and our new neutralizing antibody is a promising candidate for the prevention and treatment of EBV‐associated lymphoproliferative disorders in immunocompromised patients. Figure 1 97 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 69 Impact of Epstein‐Barr virus heterogeneity on the adaptive antiviral immune response U. Behrends1,2, J. Mautner1,2, A. A. Cirac1,2 1
2
TUM, Kinderklinik, Munich, Germany Helmholtz Zentrum Munich, AGV, Munich, Germany Background and aims Epstein‐Barr virus (EBV) is a ubiquitous γ‐herpesvirus that is associated with a number of malignancies of epithelial or lymphoid origin. Recently a new EBV strain termed M81 was isolated from an Asian nasopharyngeal carcinoma. M81 differs from the prototype EBV strains B95.8 and Ag876 in cellular tropism and lytic activity. Currently it is not known, whether the different EBV strains differ in immunogenicity and antigenicity. The aim of this project is to compare adaptive immune responses against the three EVB strains M81, B95.8, and Ag876. Methods DNA‐sequences of different EBV strains were compared to identify polymorphisms in viral proteins, respectively known T‐cell epitopes. T‐cell assays were performed to examine recognition of polymorphic peptides and to assess the role of polymorphic viral proteins in immune evasion. Western blots of viral proteins were performed to analyze cross‐reactivity in serum antibodies. T‐cell lines were generated by repeated stimulation of PBMC with lymphoblastoid cell lines (LCL) in vitro that were established by infection of B cells with B95.8 or M81 virus and characterized by FACS, ELISA, and calcein release assay. Results Although sequence variations resulted in the loss of several T‐cell epitopes in some EBV strains, LCL‐stimulated T‐cell lines showed protection against target cells infected with different viruses. Furthermore, the rate of T‐cell proliferation was increased when LCL established with M81 were used as stimulators. Conclusions Our results indicate that established EBV‐specific adaptive immunity provides cross‐protection against different viral strains. These findings have implications for adoptive T‐cell therapy and vaccine design. 98 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 70 Monoclonal antibodies as successful treatment for nosocomial infections F. Rossmann1, D. Laverde2, F. Romero‐Saavedra1, A. Kropec1, J. Huebner1 1
2
LMU, Dr. von Hauner Kinderspital, Munich, Germany Division for Infection Diseases, University Hospital, Freiburg, Germany Background Due to misuse of currently available antimicrobials and a decreased development of new antibiotics, we need to find alternative treatment options to fill the current gap of novel treatment options. The use of monoclonal antibodies (mAbs) instead of antibiotics is advantageous as it on the one hand enables prophylaxis of patients at particular risk, such as patients in intensive care units. On the other hand, development of resistance or immune evasion is very unlikely if highly conserved bacterial surface structures are targeted. Methods The complex variety of antibodies in a healthy individual was used as starting material. Therefore, B‐cells from a healthy donor showing efficient opsonic activity were immortalized with EBV. The selection process of the antibodies is based on a functional screen (i.e., uptake and killing of pathogens by phagocytes) and not ‐ as in most approaches ‐ on affinity. An opsonophagocytic assay was used and supernatants of microtiter‐wells were assessed, thereby testing hundreds of B‐cells producing different antibodies. Limited dilution was carried out to identify a small number or single B‐cells producing highly opsonic antibodies. Variable domains of light and heavy chains were subsequently cloned into a eukaryotic vector and expressed as IgG1 in CHO cells. Results We identified a set of human monoclonal antibodies that bind polysaccharide structures of different Gram‐
positive bacteria and these bacteria (three different enterococcal and 2 staphylococcal strains, including a MRSA and a VRE strain) can be subsequently eliminated by antibody‐mediated complement deposition and phagocytosis (opsonizing antibodies). Unexpectedly, all mAbs possessed the same light chain suggesting that the light chain may be crucial for carbohydrate‐antigen binding. The pattern of the variable domains showed about 95% identity to the germline configuration, confirming the importance of IgMs during the first‐line defense. Cloning of theses variable domains in front of IgG1 constant domains enabled us to produce highly potent antibodies against enterococci and staphylococci. Opsonic killing through the antibodies could be observed and protective efficacy could be confirmed in several therapeutic and prophylactic animal models. Conclusion Combining variable domains of IgMs with an IgG1‐mediated immune response resulted in efficient clearance of several Gram‐positive pathogens. Our results indicate that opsonic antibodies against conserved cell‐wall structures may be used as therapeutic or prophylactic treatment options against nosocomial pathogens. 99 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 71 Taxonomic Turmoil: the case of Enterobacter spp. S. Doijad1,2, N. B. Pati1,2, H. Ghosh1,2, Y. Yao1,2, L. Falgenhauer1,2, C. Imirzalioglu1,2, J. Overmann3,4, S. Glaeser5 T. Hain1,2, P. Kämpfer5, T. Chakraborty1,2 1
Justus‐Liebig University, Medical Microbiology, Giessen, Germany German Center for Infection Research (DZIF), Giessen, Germany 3
Leibniz Institute DSMZ‐German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany 4
German Center for Infection Research (DZIF), Braunschweig, Germany 5
Institute for Applied Microbiology, Giessen, Germany 2
The genus Enterobacter comprises of pathogenic as well as environmental strains. The taxonomic assignment of several species to the genus Enterobacter is controversial. Presently, the genus Enterobacter comprises 34 species, with several being repeatedly reclassified based on classical genome typing methods e.g. 16S rDNA or multilocus sequence typing (MLST). This has led to shifts in the classification and even assignment to new species groups. Here we explore the use of whole genome sequencing (WGS) for taxonomic classification. Sequences of 110 publicly available and in‐house sequenced Enterobacter reference strains E. kobei DSM‐
27110, E. ludwigii DSM‐16688 and E. agglomerans DSM‐3493 together with 43 strains from other closely related species of the Enterobacteriaceae family were investigated. We used Average Nucleotide Identity (ANI) and in silico DNA‐DNA hybridization estimate (isDDH) to determine the genetic relatedness among the species. The result obtained by both methods was congruent. Using reference strains as representative of a particular species, gross misclassification of 39/41 E. cloacae, 14/14 E. aerogenes, 2/2 E. asburiae and 2/2 E. hormaechei isolates, was detected. All of the E. aerogenes strains, including the reference strain, were identified as a members of the unified cluster of genus Klebsiella. In addition, E. agglomerans, E. kobei, E. massiliensis and E. lignolyticus can be considered as distant members of the genus Enterobacter. Using robust cut‐off values for species differentiation, 10 additional novel species can now be attributed to the genus Enterobacter. Our study indicates an urgent need to revise the taxonomic classification of this genus and will have huge implications in clinical and environmental microbiology. 100 Infections of the immunocompromised Host – Nosocomial and Gastrointestinal Infections P 72 Meta‐Analysis of Incidence and Mortality of Candidaemia in Europe P. Köhler1,2, O. A. Cornely3,1,2,4, D. Tacke1, M. J. G. Vehreschild1,4, H. Wisplinghoff5, J. J. Vehreschild1,4 1
Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging‐Associated Diseases, University of Cologne, Cologne, Germany 3
Clinical Trials Centre Cologne, ZKS Cologne, BMBF 01KN1106, Cologne, Germany 4
German Centre for Infection Research, Partner Site Bonn‐Cologne, Cologne, Germany 5
Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany 2
Background Candidaemia is a serious hazard to hospitalised patients, but European epidemiological data is restricted to national studies focussing on Northern Europe, population‐based surveillance programs or studies conducted in distinct local areas. Methods To determine the European incidence and mortality of candidaemia, we performed a Web of Knowledge™ search to identify European surveys, surveillance programs, as well as mono‐ and multicentre studies from 2000 to 2014. We included European studies with appropriate data on total cases, study duration, incidence and/or mortality rates. Complete datasets were categorised into population‐based and hospital‐based epidemiological studies and were analysed separately. Subgroup analysis and was performed for geographic distributions, time‐dependent development and species shifts. Results In population‐based studies, 13,368 candidaemias were diagnosed in 741,830,199 patients, revealing an overall incidence of 3.1 per 100,000 inhabitants (95% CI 2.98‐3.15). The highest incidence was observed in intensive care units (8.3/1,000 admissions [7.45‐9.06], mortality 43.1% [40.5‐45.7]), followed by tertiary care centres (1.1/1,000 admissions [0.99‐1.12], mortality 39.3% [38.1‐40.6]) and the composite cohort of teaching and general hospitals (0.42/1,000 admissions [0.41‐0.44]; mortality 34.9% [33.6‐36.3]). Over time, we observed an increase of candidaemia caused by Candida parapsilosis and Candida glabrata. While clinical cohorts show regional differences, we extrapolate a daily candidaemia incidence in Europe of 63 (95% CI 60‐64), of which 22 (95% CI 21‐23) have fatal outcome. Conclusions Incidence, species distribution and outcome of candidaemia differ considerably between clinical cohorts, European regions and over time. We observed an increasing incidence of candidaemia and an increasing proportion of Candida spp. other than C. albicans. 101 M y c o b ac teri a, T u b erc u l o si s and M al ari a
P7 3
S af ety and protective e cacy o f intravenous i m m u ni z ation w i th c ry o p reserved
Pl asm o di u m f al c i p aru m sp o ro z o i tes u nder c h em o p ro p h y l ax i s ( T Ü C H M I - 0 0 2 )
B. Mordmüller1, S. Borrmann1, S. off ann2, P . Kremsner1,2
1
2
Institut für Tropenmedizin, niversit t Tübingen, Tübingen, Germany
Sanaria Inc, Rockville, USA
posure to 15 P. falciparum (P f)-infected mos uitoes, administered t ree times at ont ly intervals under
continuous c e oprop ylaxis wit c loro uine is ig ly e cacious in preventing ase ual blood stage infection
following subse uent C ontrolled Human Malaria Infection (C HMI) in ealt y, adult, malaria-naï ve individuals.
To translate and expand t is i
uni ation strategy, we replaced mos uito bites wit a p ar aceutical product
t at can be easily administered in exact doses.
In t e first C HMI-trial (TÜ C HMI-001) we establis ed a ig ly standardized intravenous regimen of aseptic,
purified, cryopreserved P f sporozoites (P fSP Z allenge) to consistently infect all malaria-naï ve volunteers. In
our second trial (TÜ C HMI-002), an immuni ation sc edule t at is practicable and results in ig -grade
protection against malaria was successfully developed.
ROBUST
STARKE WIRKSAMKEIT 1–5
HOHER SCHUTZ VOR RESISTENZEN 3,5,6
(atazanavir)
STARK. ROBUST. BEWÄHRT.
1,5,7
1. Molina JM et al. Lancet 2008;372(9639):646–655. 2. Daar ES
et al. Ann Intern Med 2011;154(7):445–456. 3. Rockstroh JK et al.
J Acquir Immune Defic Syndr 2013;62(5):483–486. 4. Lennox JL
et al. Ann Intern Med 2014;161(7):461–471. 5. DeJesus E et al.
Lancet 2012;379(9835):2429–2438. 6. Molina JM and the CASTLE Study Team. J Acquir Immune Defic Syndr 2010;53(3):323–
332. 7. REYATAZ® Fachinformation, Stand April 2014.
REYATAZ® 100 mg / 150 mg / 200 mg / 300 mg Hartkapseln.
Wirkstoff: Atazanavir. Zusammensetzung: 100 mg / 150 mg /
200 mg / 300 mg Atazanavir pro Kapsel. Sonstige Bestandteile:
REYATAZ® Hartkapsel: Crospovidon, Lactose-Monohydrat, Magnesiumstearat. Kapselhülle und Drucktinte: Gelatine, Indigocarmin
(E132), Titandioxid (E171), Schellack, Ammoniumhydroxid, Propylenglycol, Simeticon. Anwendungsgebiete: Zur antiretroviralen
Kombinationsbehandlung von HIV-1-infizierten Erwachsenen und
Kindern ab 6 Jahren mit einem Körpergewicht von mindestens
15 kg. Gegenanzeigen: Überempfindlichkeit gegenüber Atazanavir oder einem der sonstigen Bestandteile. Patienten mit mäßiger bis schwerer Leberinsuffizienz. Nicht gleichzeitig anwenden
mit Arzneimitteln, die Johanniskraut enthalten. Die gleichzeitige
Anwendung von REYATAZ® mit Simvastatin oder Lovastatin ist
kontraindiziert. Nicht gleichzeitig anwenden mit PDE5-Inhibitor
Sildenafil zur Therapie der pulmonalen arteriellen Hypertonie. In
Kombination mit Ritonavir nicht gleichzeitig anwenden mit Rifampicin und mit Arzneimitteln, die Substrate der Cytochrom- P450Isoform CYP3A4 sind und eine geringe therapeutische Breite
haben (z. B. Quetiapin, Alfuzosin, Astemizol, Terfenadin, Cisaprid,
Pimozid, Chinidin, Bepridil, Triazolam, oral angewendetes Midazolam, Mutterkorn-Alkaloide, insbesondere Ergotamin, Dihydroergotamin, Ergometrin, Methylergometrin). Nebenwirkungen: Bei
HIV-Patienten mit schwerem Immundefekt ist ein Immun-Reaktivierungs-Syndrom möglich. Bei HIV-Patienten mit fortgeschrittener
HIV-Erkrankung oder antiretroviraler Langzeittherapie wurden
Fälle von Osteonekrose beschrieben. Bei Patienten mit Hämophilie
Typ A oder Typ B sind vermehrt Blutungen möglich. In den ersten
drei Wochen der Behandlung können Hautausschläge (leichte mit
mäßige makulopapulöse Exantheme) auftreten. Häufig (mind.
1/100 Pat.): Kopfschmerzen, Ikterus der Augen, Erbrechen, Diarrhoe, Bauchschmerzen, Übelkeit, Dyspepsie, Ausschlag, Lipodystrophie-Syndrom, Erschöpfung, Ikterus. Gelegentlich (mind.
1/1.000 Pat.): periphere Neuropathie, Synkope, Amnesie, Schwindel, Benommenheit, Dysgeusie, Dyspnoe, Gallensteine, Pankreatitis, Gastritis, aufgeblähtes Abdomen, aphthöse Stomatitis, Blähungen, Mundtrockenheit, Nephrolithiasis, interstitielle Nephritis,
Hämaturie, Proteinurie, Pollakisurie, Urticaria, Alopezie, Juckreiz,
Erythema multiforme, toxisches Exanthem, Angioödem, DRESSSyndrom, Muskelatrophie, Arthralgie, Myalgie, Gewichtsabnahme,
Gewichtszunahme, Anorexie, gesteigerter Appetit, Bluthochdruck,
Brustschmerz, Unwohlsein, Fieber, Asthenie, allergische Reaktion,
Hepatitis, Gynäkomastie, Depressionen, Orientierungslosigkeit,
Angst, Schlaflosigkeit, Schlafstörungen, abnormale Träume. Selten (mind. 1/10.000 Pat.): Ödem, Palpitation, Nierenschmerzen,
Stevens-Johnson-Syndrom, vesikulobullöser Ausschlag, Ekzem,
Gefäßerweiterung, Myopathie, abnormaler Gang, Hepatosplenomegalie. Nicht bekannte Häufigkeit: Autoimmunerkrankungen
(wie z.B. Morbus Basedow), Torsades de pointes, QT-Verlängerung,
Diabetes mellitus, Hyperglykämie, Gallenblasenfunktionsstörungen. Weitere Warn- und Behandlungshinweise siehe Fachinformation. Verschreibungsstatus: Verschreibungspflichtig. Stand des
Textes: August 2014. Pharmazeutischer Unternehmer: Bristol-Myers Squibb Pharma EEIG, Uxbridge Business Park, Sanderson Road, UB8 1DH Uxbridge, Middlesex, Vereinigtes Königreich.
687DE14PR10130-01
STARK
BEWÄHRT
Adult, ealt y, malaria-naï ve volunteers were exposed to t e P fSP Z-e uivalent of t ree times 5, 20 and 8 0
os uito bites. All volunteers received 10 weekly doses of c loro uine and t ree inj ections by direct venous
inoculation (DVI) of eit er placebo (n= 15), 3,200 P fSP Z (n= 9 ), 12,8 00 P fSP Z (n= 9 ) or 51,200 P fSP Z (n= 9 ). P fSP Z
in ections and c e oprop ylaxis were well tolerated and safe. All volunteers of t e 51,200 P fSP Z-group were
protected from subse uent controlled uman malaria infection (C HMI). In t e second stage of trial, rapid
i
unization regimens using 51,200 P fSP Z and combination c emoprop ylaxis wit azit ro ycin were tested.
e study is ongoing and results of i
unological analyses of bot trial stages will be presented.
Mycobacteria, Tuberculosis and Malaria P 74 Plasmodium falciparum infection in febrile Congolese children: prevalence of clinical malaria and influence of sickle cell trait M. K. Etoka‐Beka1,2, M. Kombo1, Julia D.t3, P. Poulain1,4,5,6,7, C. Vouvoungui1, F.Koukouikila‐Koussounda1,2 F. Ntoumi1,2,3 1
Fondation Congolaise pour la Recherche Médicale, Faculté des Sciences de la Santé, Marien Ngouabi University, Brazzaville, Republic of Congo 2
Faculté des Sciences et Techniques, Marien Ngouabi University, Brazzaville, Republic of Congo 3
Institute for Tropical Medicine, University of Tübingen, Tübingen, Germany 4
Institut National de la Santé et de la Recherche Médicale U 1134, Paris, France 5
UMR_S 1134, DSIMB, Université Paris Diderot, Sorbonne Paris Cite, Paris, France 6
Institut National de la Transfusion Sanguine, DSIMB, Paris, France 7
UMR_S 1134, DSIMB, Laboratory of Excellence GR‐Ex, Paris, France Background Even though recent data have shown a reduction in malaria prevalence in symptomatic patients in the Republic of Congo, malaria remains a public health problem in the country. The introduction of tools like insecticide treated bed nets and artemisin‐based combination therapies contributed significantly to this reduction and it was of interest to determine the current part of malaria in children harboring fever and consulted in a pediatric hospital of Brazzaville. As a second objective, we investigated whether the sickle cell trait carriage (18% of the Congolese population) influenced the proportion of children with clinical malaria. Methods Blood samples of children aged <10 years with an axillary temperature ≥ 37.5°C were collected, thick and thin blood smears prepared and read by microscopy. All samples were screened for the presence of Plasmodium falciparum (P. falciparum) by nested PCR using the P. falciparum MSP2 marker to detect submicroscopic infections and to determine the allelic diversity as well as the multiplicity of infection (MOI). Sickle cell trait was screened using polymerase chain reaction (PCR) method. Results A total of 229 children with fever were recruited from September 2014 to February 2015. Among them, 9.6% had detectable parasites by microscopy and 21.4% had a submicroscopic infection (positives by PCR and negative by microscopy). Thirty (13.1%) children had the sickle cell trait (HbAS) and 199 (86.9%) had normal hemoglobin (HbAA). Clinical malaria was diagnosed in 9.1% and 11.3% of HbAA and HbAS groups, respectively. Submicroscopic infection was detected in 20.6% and 26.7% for HbAA and HbAS children respectively (p‐value > 0.05). The MOI was 1.63 and 2.21 for HbAA and HbAS children, respectively. Conclusions This study reports a moderate prevalence of malaria in febrile children. It also shows no influence of sickle cell trait on clinical malaria in Congolese children. Key words Sickle cell trait; Plasmodium falciparum; fever; submicroscopic infection; children; Congo 103 Mycobacteria, Tuberculosis and Malaria P 75 Characterization of Plasmodium falciparum asymptomatic infection in pregnant women from the Republic of Congo F. Koukouikila‐Koussounda1, F. Ntoumi1,2, D. Bakoua1, A. Fesser1, M. Kombo1, J. C. Vouvoungui1 1
Fondation Congolaise pour la Recherche Médicale, Laboratoire de Biologie Moléculaire (Molecular Biology laboratory), Brazzaville, Congo 2
Institute of Tropical Medicine, University of Tübingen, Tübingen, Germany Background Malaria in pregnancy remains a serious public health problem in the Republic of Congo despite the implementation of intermittent preventive treatment with sulfadoxine‐pyrimethamine in 2006. The aim of this cross‐sectional study was to characterize Plasmodium falciparum infections and determine its prevalence and possible risk factors in pregnant Congolese women attending an antenatal clinic in a periurban area of Brazzaville. Methods This study was conducted from March 2012 to December 2013 at Madibou health centre in a Southern district of Brazzaville where several years ago, high malaria resistance to sulfadoxine‐pyrimethamine (SP) was reported. Pregnant women were enrolled during antenatal visits and the number of received SP doses for intermittent preventive treatment during pregnancy (IPTp) was recorded as well as individual sociodemographic data. Peripheral blood was collected and P. falciparum infection was checked using microscopy and PCR. Haemoglobin concentration was measured and P.falciparum positive samples were typed for merozoite surface protein 2 (msp2) allelic diversity. Results A total of 363 pregnant women were recruited. The prevalence of asymptomatic P. falciparum infection was 7% and 19% by microscopy and by PCR, respectively. More than one half (51.5%) of the pregnant women were anaemic. Multivariate analysis indicated that P. falciparum infection was associated with anaemia. It was also observed that women who have received 2 or 3 doses IPTp‐SP have significantly lower prevalence of infection. The typing of isolates based on msp2 polymorphic marker revealed that clonality and multiplicity of infection (MOI) were comparable across all the studied status. Conclusions This first study investigating asymptomatic malaria infection on pregnant women of the Republic of Congo shows that sub‐microscopic infections is clearly associated with maternal anaemia, and use of IPTp‐SP is associated with reduced prevalence of infection. Keywords Plasmodium falciparum, Pregnant women, msp2, Congo Brazzaville 104 Mycobacteria, Tuberculosis and Malaria P 76 Prevalence of Influenza Subtype A and B in hospitalised children with severe febrile illness in rural Ghana B. Hogan1, L. Ammer1, N. Sarpong2, T. Binger2, D. Eibach1, R. Krumkamp1, D. Dekker1, A. Jaeger1 Y. Adu‐Sarkodie3, E. Owusu‐Dabo2, J. May1 1
Bernhard‐Nocht‐Institut für Tropenmedizin Hamburg, Infektionsepidemiologie, Hamburg, Germany Kumasi Centre for Collaborative Research, Kumasi, Ghana 3
Kwame Nkrumah University of Science and Technology, Kumasi, Ghana 2
Background In many sub‐Saharan countries the identification of pathogens causing febrile illness in children is challenged by the absence of appropriate diagnostic facilities. Thus, the spectrum and relevance of infections other than malaria and malaria co‐infections remains unclear. In this study we aim to investigate the prevalence of Influenza Subtype A/B among hospitalised children from rural Ghana. Methods Children aged ≤15 years with fever ≥ 38,0°C were recruited when admitted to the children’s ward of the Agogo Presbyterian Hospital in the Ashanti region, Ghana. An oro‐pharyngeal swab was taken from all children and RNA was extracted. Screening of samples for Influenza A and B was done by real‐time PCR and positive Influenza A samples were further differentiated for H1N1pan2009 and H3N2 following a real‐time RT‐PCR protocol. Influenza cases were described by age and time of disease onset. Children were tested for malaria parasites by thick and thin film. Results From january 2014 to april 2015 922 children with severe febrile illness were recruited for the study. Median age at hospital attendance was three years (interquartile range: 1‐5) and proportion of females 45.1%. Influenza Type A was prevalent in 20 samples (2.2%) and Type B in 26 samples (2.8%) while Influenza A viruses of the pandemic H1N1 subtype were found in 12 samples and the H3N2 subtype in 6. Malaria parasites were found in 54.0% of the study population and a Malaria/Influenza co‐infection in 4 (0.3%) patients. Signs of acute respiratory infection were present in 10 (55.6%) Influenza subtype A and in 12 (48.1%) Influenza Type B cases. Influenza Type B was predominant during the rainy season in June 2014 while Type A dominated one year later. Conclusion First results of this ongoing study suggest that in a malaria endemic region a small proportion of severe febrile infections in children could be explained by Influenza A/B, while malaria/influenza co‐infections seem to be rare. Raised awareness among healthcare staff that Influenza could be a differential diagnosis in malaria endemic regions may improve the clinical management of patients. 105 Mycobacteria, Tuberculosis and Malaria P 77 An atypical radiological presentation of pulmonary Mycobacterium intracellulare infection H. J. F. Salzer1, N. Wassilew1, C. Lange1, G. Günther2 1
2
Forschungszentrum Borstel, Klinische Infektiologie, Borstel, Germany School of Medicine, University of Namibia, Department of Medicine, Windhoek, Namibia Introduction Pulmonary infections by nontuberculous mycobacteria (NTM) usually affect patients with chronic lung diseases, but it has been also described in healthy individuals. Two major radiological patterns have been described so far including the fibrocavitary form and the nodular/bronchiectatic one. We present an atypical radiological presentation of pulmonary Mycobacterium intracellulare infection. Clinical presentation A 77‐year‐old woman presented with a 3‐month history of chronic cough, shortness of breath and fatigue. There was no history of previous lung disease. Physical examination and basic laboratory testing were completely unremarkable. Computed tomography (CT) of the chest revealed multiple small nodules strictly limited to the basal segments of the right lower lobe with tree‐in‐bud sign, mild bronchiectasis (Figure 1) and with reactive mediastinal, and hilar lymphadenopathy (Figure 2, arrow). Histopathological analysis of a transbronchial biopsy of the right lower lobe showed multiple non‐caseating, epithelioid cell granulomas and Langerhans giant cells. Endobronchial ultrasound guided needle aspiration of an enlarged hilar lymph node showed similar results. These findings suggested a possible diagnosis of mycobacterial infection, however, repeated smears for acid‐fast bacilli as well as polymerase‐chain‐reaction assay testing (GeneXpert® MTB/RIF, Cepheid, Sunnyvale, CA, USA) for Mycobacterium tuberculosis from three different sputum samples were negative. After six to eight days of incubation all samples collected were culture‐positive for non‐tuberculous mycobacteria. Further molecular genetic identification (GenoType Mycobacterium CM/AS, Hain Lifesience, Nehren, Germany revealed Mycobacterium intracellulare. After 16 month of antimycobacterial treatment with clarithromycin 500 mg twice a day, ethambutol 1000 mg once per day, and rifabutin 300 mg once per day repeated CT of the chest showed no radiological improvement, while patient’s symptoms resolved. Treatment was stopped and the patient followed up after three months without clinical or radiological deterioration. Conclusion Pulmonary NTM infection can present a wide variety of radiological presentations. Therefore pulmonary NTM infection should be kept in mind in previously healthy individuals without a history of lung disease presenting with multiple nodules without significant bronchiectasis. Figure 1 Figure 2 106 Mycobacteria, Tuberculosis and Malaria P 78 Severe skin and soft tissue infection caused by Mycobacterium abscessus subsp. abscessus in an immunocompromised woman H. J. F. Salzer1, E. Terhalle1, N. Wassilew1, C. Herzmann1, C. Lange1 1
Forschungszentrum Borstel, Klinische Infektiologie, Borstel, Germany Introduction Infections caused by mycobacterium abscessus complex includes a group of fast growing, often multidrug‐
resistant, nontuberculous mycobacteria that are responsible for a wide spectrum of diseases including skin and soft tissue infections. We report a severe skin and soft tissue infection caused by Mycobacterium (M.) abscessus subsp. abscessus in an immunocompromised woman. Clinical presentation A 73‐year‐old woman presented with multiple ulcerative skin and soft tissue lesions of her right lower leg. 10‐
month prior immunosuppressive treatment with prednisolone 7 mg once per day and azathioprine 50 mg twice a day was initiated due to active polymyalgia rheumatica. Shortly after she noticed small subcutaneous nodules pretibial. Topic treatment with cortison‐containing ointment and empiric treatment with clarithromycin showed no effect. Six month later the number of nodules increased significantly. Nodules were excised recurrently, however, no wound closure was achieved. Mycobacterial analyses of subcutaneous tissue revealed M. abscessus subsp. abscessus. Real time polymerase chain reaction assay detected inducible clarithromycin resistance (erm‐gen). Computed tomography of the chest showed no evidence for a pulmonary manifestation and sputum smears were repeatedly negative for acid‐fast bacilli. We initiated antimycobacterial treatment with doxycyline 200 mg once per day, azithromycin 500 mg once per day, and bedaquiline 400 mg once per day for 14 days followed by 200 mg three‐times a week. After 35 days skin lesions improved significantly and treatment was continued. Conclusion Clinical management of skin and soft tissue infections caused by M. abscessus subsp. abscessus includes often the use of multiple antiinfective agents for several months and the potential use of adjunctive surgery. Figure 1 Figure 2 107 Mycobacteria, Tuberculosis and Malaria P 79 Virulent and Avirulent Strains of M. avium hominissuis regulate Neutrophil Functional Capacities Differentially by Inhibition of p38MAPK A. S. Neu1, A. Nowag1, E. van Gumpel1, S. Winter1, J. Rybniker1, G. Plum2, P. Hartmann1,2 1
2
Uniklinik Köln, Medizinische Klinik 1, Exp. Infektiologie, Cologne, Germany Uniklinik Köln, Medizinische Mikrobiologie, Immunologie und Hygiene, Cologne, Germany Introduction Neutrophils (PMNs), in addition to monocytes and macrophages (MΦs), have been implicated in the innate immune response during the initial phase of mycobacterial infection. However, the available knowledge on their role in mycobacterial infection is confounding. Objectives We investigated the cell‐autonomous capacities of human PMNs during mycobacterial infection to control mycobacteria. Methods Human PMNs were infected with two clinical isolates of M. avium hominissuis, which had been previously defined as virulent and avirulent based on their generation time in human macrophages. The capacities investigated were phagocytosis, degranulation, production of reactive oxygen species (ROS), secretion of cytokines, formation of neutrophil extracellular traps (NETs) by the release of nuclear chromatin, phagosomal processing and killing capacity. Most of those processes are dependent on the p38MAPK. Results Though both strains were phagocytized by the PMNs, we could detect, that TLR2 receptor, necessary for internalization of the mycobacteria, is lower expressed on cell surface of PMNs infected with the virulent strain in comparison to those infected with avirulent strain. However, phagocytized mycobacteria were not efficiently killed by PMNs, as they blocked phagosomal processing. Remarkable, activation of the central „keyplayer“ of many processes, p38MAPK, was differentially regulated, as virulent strain down regulated p38MAPK almost entirely for up to 60min post infection. And in fact, p38MAPK dependent processes such as degranulation and release of ROS as well as of IL‐8 were significantly suppressed upon infection of PMNs with the virulent strain compared to the avirulent strain. Also the formation of NETs, a process being considered as ROS dependent, was significantly less induced upon infection with the virulent strain when compared to the avirulent. Interestingly, NETs of mycobacteria infected PMNs revealed higher concentrations of Lactoferrin, a glycoprotein with antimycobacterial activity, than those stimulated with non‐bacterial positive control. Surprisingly, neither of the two strains induced significant apoptosis nor necrosis in PMNs compared to uninfected cells. Co‐infection experiments of mycobacteria infected MΦs together with non‐infected PMNs revealed, that addition of PMNs is able to promote the killing capacity of the macrophages. Conclusion Both clinical isolates of M. avium hominissuis applied in this study escape early host defense by blocking of phagosome maturation of PMNs independently from their virulence. However, virulence impacts on the differential regulation of p38MAPK linked processes such as degranulation, ROS, release of pro‐inflammatory IL8 and formation of NETs. We propose that these processes are meaningful for the granuloma microenvironment as they support the MΦs to kill the internalized mycobacteria. 108 Mycobacteria, Tuberculosis and Malaria P 80 Epidemiology of pyrazinamide resistant tuberculosis in Africa ‐ a multicenter study in seven high incidence countries P. Beckert1,2, A. Rachow3,4, E. Sanchez‐Padilla5, S. Ali6, E. Bruske7,8, A. Alabi9, B. Lell9, K. Azam10, C. Bila10 S. Pazia Juma11,12, D. Mapamba13, L. Isherwood14, I. Sanne15, N. E. Ntinginya13, G. Kibiki11, M. Bates16, S. Viegas10 M. Frank7,8, M. Bonnet5, S. Niemann1,2, M. Hölscher3,4 1
Research Center Borstel, Molecular Mycobacteriology, Borstel, Germany German Center for Infection Research, Partner Site Hamburg‐Borstel‐Lübeck, Hamburg‐Borstel‐Lübeck, Germany 3
University of Munich, Department of Infectious Diseases & Tropical Medicine, Munich, Germany 4
German Center for Infection Research, Partner Site Munich, Munich, Germany 5
Epicentre, Paris, France 6
Jimma University, Jimma, Ethiopia 7
University of Tuebingen, Institute for Tropical Medicine, Tuebingen, Germany 8
German Center for Infection Research, Partner Site Tuebingen, Tuebingen, Germany 9
Albert Schweitzer Hospital, Centre de Recherches Médicales de Lambaréné (CERMEL), Lambaréné, Gabon 10
Ministério da Saúde, Instituto Nacional de Saúde, Maputo, Mozambique 11
Kilimanjaro Christian Medical Center, Moshi, Tanzania 12
Kilimanjaro Clinical Research Institute, Moshi, Tanzania 13
Mbeya Medical Research Center, Mbeya, Tanzania 14
National Priority Programme, National Health Laboratory Service, Johannesburg, South Africa 15
University of the Witwatersrand, Johannesburg, South Africa 16
UNZA‐UCLMS Research & Training Programme, Lusaka, Zambia 2
According to the World Health Organization an estimated number of 2.8 million people were newly infected by tuberculosis (TB) and 690,000 died from TB in Africa in 2013. Even more worrisome is the emergence of drug resistant Mycobacterium tuberculosis complex (MTBC) isolates in Africa. Pyrazinamide (PZA) remains an essential TB drug and will be a key component of new combination treatment regimens set to be assessed in several upcoming clinical trials of TB treatment. As such, developing the capability to perform reliable and rapid PZA drug susceptibility testing (DST) has become a high priority. The project aims for describing the epidemiology of PZA resistance at seven African trial sites, phenotypic and genotypic data will be correlated to support the development of new and rapid PZA resistance tests. To get an insight in transmission dynamics of TB at the seven sites, we analysed the population structure of the MTBC applying molecular typing methods (spacer oligonucleotide typing (spoligotyping) and 24‐loci mycobacterial interspersed repetitive units ‐ variable number of tandem repeats (MIRU‐VNTR) typing). These methods are ideally suited to analyze the population structure and get a first insight into chains of transmission of MTBC isolates. So far 770 African MTBC isolates from seven countries, Swaziland (275), Ethiopia (109), Tanzania (129), Gabon (107), Mozambique (62), Zambia (58) and South Africa (36) have been analysed.The first analysis revealed that 23% of all analysed isolates are resistant to PZA. Most of the PZA resistant isolates (69%) originate from Swaziland. Further sequence analysis of pncA, the gene in which most of the PZA resistance mediating mutations occur, showed a large variety of different mutations dispersed all over the gene.With the help of molecular genotyping we were able to identify several different genotypes spreading at the seven investigated sites. The major genotypes present in this study are the Latin‐American Mediterranean (20%), X‐type (12%), Haarlem (10%), S‐type (9%) and Beijing (8%). Analysis of molecular clustering revealed that 53% of the isolates are clustered in 89 clusters; the largest cluster comprised 37 isolates originating from Swaziland, South Africa and Mozambique. In conclusion, our study identified a diverse level of PZA resistance at the different African sites. In addition the resistance is not mediate by one particular mutation in pncA rather than by several mutations. This diversity of mutations complicates the development of an easy‐to‐use molecular test to diagnose patients infected with PZA‐resistant TB in Africa.The molecular genotyping showed that in contrast to Asia and Eastern Europe, the TB epidemic in Africa is driven by a variety of genotypes. 109 Mycobacteria, Tuberculosis and Malaria P 81 Geographical mapping of questions on mycobacterial infections addressed to the consulting service of the Research Center Borstel I. D. Olaru1,2, B. Kalsdorf1,2, J. Heyckendorf1,2, N. Wassilew1,2, E. Terhalle1, F. Brinkmann3, F. Ahrens4 H. von Bernuth5, C. Lange1,2 1
Research Center Borstel, Division of Clinical Infectious Diseases, Borstel, Germany German Center for Infection Research, Clinical Tuberculosis Center Borstel, Borstel, Germany 3
University Children's Hospital, Ruhr‐ University Bochum, Department of Paediatric Pneumology, Bochum, Germany 4
Children's Hospital "Altona", Hamburg, Germany 5
Charité University Medicine, Pediatric Pneumology and Immunology, Berlin, Germany 2
Background Mycobacterial infections continue to cause significant morbidity and mortality. According to the Robert‐Koch Institute over 4300 cases of tuberculosis (TB) were reported in 2013 in Germany. Because it is not reportable, the number of patients with non‐tuberculous mycobacterial (NTM) disease is unknown. Within the German Center for Infection Research a consulting service was founded to help answer physicians’ questions concerning mycobacterial diseases. This comprises a team of experts in the fields and trainees in infectious diseases. The questions are addressed via telephone calls and letters while complex cases can be uploaded on an online platform or are discussed during expert meetings. Objective To evaluate the questions addressed to the consulting service from the Research Clinic Borstel with regards of the topic and the geographical location from where they were addressed. Methods The content and location of origin of the questions addressed to the consulting physicians from the Research Center Borstel between April and August 2015 were analysed. The geographical location was determined using the prefix of the telephone number and the information given during the call. The corresponding postal code and the GPS coordinates were identified. Epi Info v.7 (CDC, USA) was used for data collection, analysis and geographical mapping. Results Of the 312 consulting requests addressed during the study period, 230 (73.7%) were concerning TB and 82 (26.3%) NTM infections. There were 28 questions regarding patients with suspected or confirmed multidrug‐resistant TB. Sixty‐six (21.2%) questions were regarding the diagnosis of mycobacterial infections (50 for TB and 16 for NTM), 187 (59.9%) about therapy (123 for TB and 64 for NTM), 20 (6.4%) about the management of drug‐induced adverse events, 26 (8.3%) about preventive chemotherapy for TB and 13 (4.2%) on the risk of transmission of TB. Most questions were addressed by physicians from North Rhine‐Westphalia, 102 (32.7%), followed by Baden‐Württenberg 45 (14.4%), Hessen 40 (12.8%), Lower Saxony 33 (10.6%), and Bavaria 24 (7.7%). Sixty‐eight (21.8%) of the questions were from the other German federal states or from other countries. The geographical location and the type of mycobacterial infection are shown in Figure. Conclusions A large number of questions regarding mycobacterial infections, including a relatively large number of questions on multidrug‐resistant TB, are addressed to the consulting service of the Research Center Borstel. Most questions originated from the western and southern Figure 1 regions of Germany. 110 Mycobacteria, Tuberculosis and Malaria P 82 The German DZIF M/XDR Cohort J. Heyckendorf1, I. D. Olaru1, B. Kalsdorf1, N. Wassilew1, C. Ehlers1, N. Smitsman1, J. Hofmeister1,L. Busenbender1 P. Sanchez‐Carballo1, C. Lange1 1
FZ Borstel, Klinische Infektiologie, Borstel, Germany Background Tuberculosis (TB) is responsible for approximately 1.5 Million deaths annually. Still, the emergence of drug‐
resistant TB is alarming. Globally, about 5% of all TB cases and 20,5% among previously treated patients are infected with multi‐drug‐resistant (MDR) M. tuberculosis (MTB) strains. The hot spots of MDR‐TB are found in Eastern European countries and in Central Asia. Although the standard recommended treatment length for the therapy of susceptible TB is 6 months, the World Health Organization recommends a duration of 20 months for the treatment of patients with MDR‐TB and extensively drug‐resistant (XDR) TB. The optimal duration for the treatment of TB likely differs between individuals and depends on a variety of variables, such as the extent of the disease, the immune status of the host, and the virulence and the drug resistance of the causative strain of MTB. Some patients with M/XDR‐TB may have to be treated for more than 20 months, but shorter treatment durations may be possible to achieve cure for the majority of patients with M/XDR‐TB. Personalization of the duration of treatment for TB, especially for patients with M/XDR‐TB, is highly desired (Heyckendof J. et al. AJRCCM 2014). Objective The DZIF M/XDR TB cohorts’ aim is the identification of surrogate biomarkers for treatment outcome prediction (i.e. cure or treatment failure) in patients with M/XDR‐TB in order to individualize the duration of therapy. Methods Since March 2013, patients with M/XDR TB have been enrolled at 5 clinical centers in Germany. Recruitment is ongoing. Besides clinical and microbiological parameters, host biomarker material is obtained at fixed and at variable clinical milestones (smear conversion and culture conversion). Results A total number of 95 patients were recruited between March 2013 and August 2015 of witch 59 were infected with susceptible MTB strains and 36 with M/XDR‐MTB strains. Interim analysis shows microbiological, clinical and biomarker kinetics in the course of early and late treatment. Mean time to smear conversion in non‐
M/XDR was 52.76 days vs. 94.35 days in M/XDR TB and mean time to culture conversion in non‐M/XDR was 53.07 days vs. 96.11 days in M/XDR TB. The radiological extent of disease before treatment initiation measured by the Ralph score was similar in both groups (non‐M/XDR 64.47 vs. 62,65 M/XDR), whereas age (non‐M/XDR 48.98 vs. 38.83 M/XDR) and disease severity (non‐M/XDR 3.46 vs. 4.87 M/XDR) measured by a clinical scoring system differed. Currently, large‐scale candidate biomarker analysis is conducted. Conclusions The evaluation of surrogate biomarkers can lead to the individualization of TB therapy in the future. Ongoing patient recruitment in Germany and future recruitment at the DZIF study site in Bucharest (Romania) will serve to validate the identified markers and finally lead to individualized biomarker‐guided M/XDR‐TB therapy durations. 111 Mycobacteria, Tuberculosis and Malaria P 83 Griselimycins: Streptomyces‐derived leads with potent antituberculosis activity that target the sliding clamp A. Kling1,2, P. Lukat1,3,2, N. Zaburannyi1,2, J. Herrmann1,2, S. Wenzel1,2, A. Bauer4, E. Fontaine5, M. Brönstrup3,4 L. Fraisse5, J. Grosset6,7, S. Lagrange5, R. Müller1,2 1
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken, Germany German Centre for Infection Research (DZIF), Partner Site Hannover‐Braunschweig, Hannover, Germany 3
Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany 4
Sanofi‐Aventis R&D, LGCR/Chemistry, Frankfurt am Main, Germany 5
Sanofi‐Aventis R&D, Infectious Disease TSU, Toulouse, France 6
Center for TB Research, Johns Hopkins University School of Medicine, Baltimore, USA 7
KwaZulu‐Natal Research Institute for TB and HIV (K‐RITH), Durban, South Africa 2
Background Griselimycins (GMs) are cyclic peptides derived from Streptomyces strains. GM and its optimized derivatives are highly active against sensitive and drug‐resistant M. tuberculosis, both in vitro and in vivo. Examination of the GM biosynthetic gene cluster in Streptomyces sp. revealed an additional DNA polymerase sliding clamp (DnaN), that was later found to mediate self‐resistance in Streptomyces. To inform future therapeutic strategies, the mechanism of action and the mechanism of resistance of GMs were examined in Mycobacteria. Methods The suspected self‐resistance determinant of S. caelicus was examined via over‐expression in GM sensitive S. coelicolor. Bacterial sliding clamps were heterologously expressed in E. coli and binding of GMs to sliding clamps was characterized by surface plasmon resonance and x‐ray crystallography. Resistant Mycobacteria were selected and characterized by whole genome sequencing. Expression analyses were performed using qPCR. Results GMs are active against drug‐resistant Mycobacteria, with a MIC of CGM of 0.06 μg/mL against M. tuberculosis and 0.6 μg/mL against M. smegmatis. GM derivatives are specifically active against species of the Corynebacterineae suborder. GMs demonstrated binding to the protein binding site of the bacterial DNA polymerase sliding clamp, which also binds enzymes and factors involved in replication and repair. No binding was observed to the human homolog (PCNA). Resistant Mycobacteria showed an amplified genomic region containing the dnaN gene that encodes the DNA Polymerase sliding clamp, along with the ori site. GM resistant M. smegmatis mutants developed at a very low frequency of resistance (5 × 10‐10 at 10 μg/mL GM). Investigation of the GM biosynthetic gene cluster of producing Streptomyces revealed an additional homolog of the sliding clamp, overexpression of which conferred resistance to GM in sensitive Streptomyces. Conclusions GMs are narrow spectrum agents that inhibit M. tuberculosis by binding to the peptide binding site of DnaN and by interference with binding of the DNA polymerase. Self‐resistance to GM is mediated by an additional DnaN homolog. Resistance to GM in mycobacteria occurs with low frequency and is associated with amplification of DnaN. 112 Mycobacteria, Tuberculosis and Malaria P 84 A novel reading scheme for assessing the extent of radiographic abnormalities and its association with disease severity in sputum smear‐positive tuberculosis: an observational study in Hyderabad/India Z. Grozdanovic1, L. C. Berrocal Almanza2, S. Goyal3, V. Valluri4, N. Ahmed5, R. R. Schumann3, G. Sumanlatha4 B. Lala6, H. Slevogt2 1
Charite Universitätsmedizin Berlin, Department of Radiology, Berlin, Germany University Hospital Jena, Septomics Research Centre, Jena, Germany 3
Charite Universitätsmedizin Berlin, Department of Microbiology and Hygiene, Berlin, Germany 4
Bhagwan Mahavir Medical Research Center, Department of Immunology, Hyderabad, India 5
University of Hyderabad, Department of Biotechnology and Bioinformatics, Hyderabad, India 6
Charite Universitätsmedizin Berlin, Department of Pediatric Radiology, Berlin, Germany 2
Background Existing reading schemes for chest X‐ray (CXR) used to grade the extent of disease severity at diagnosis in patients with pulmonary tuberculosis (PTB) are often based on numerical scores that summate specific radiographic features. However, since PTB is known to exhibit a wide heterogeneity in pathology, certain features might be differentially associated with clinical parameters of disease severity. Objective We aimed to grade disease severity in PTB patients at diagnosis and after completion of DOTS treatment by developing a reading scheme based on five different radiographic manifestations and analyze their association with the clinical parameters of systemic involvement and infectivity. Methods 141 HIV‐negative adults with newly diagnosed sputum smear‐positive PTB were enrolled in a prospective observational study in Hyderabad, India. The presence and extent on CXRs of five radiographic manifestations, i.e., lung involvement, alveolar infiltration, cavitation, lymphadenopathy and pleural effusion, were classified using the new reading scheme by using a four‐quadrant approach. We evaluated the inter‐reader reliability of each manifestation, and its association with BMI and sputum smear positivity at diagnosis. The presence and extent of these radiographic manifestations were further compared with CXRs on completion of DOTS treatment. Results At diagnosis, an average lung area of 51.7% +/‐ 23.3% was affected by radiographic abnormalities. 94% of the patients had alveolar infiltrates, with 89.4% located in the upper quadrants, suggesting post primary PTB and in 34.8% of patients cavities were found. We further showed that the extent of affected lung area was a negative predictor of BMI (β value ‐0.035, p 0.019). No significant association of BMI with any of the other CXR features was found. The extent of alveolar infiltrates, along with the presence of cavitation, were strongly associated with sputum smear positivity. The microbiological cure rate in our cohort after 6 months of DOTS treatment was 95%. The extent of the affected lung area in these patients decreased from 56.0% +/‐ 21.5% to 31.0 +/‐ 20% and a decrease was also observed in the extent of alveolar infiltrates from 98.4% to 25.8% in at least one quadrant, presence of cavities from 34.8% to 1.6%, lymphadenopathy from 46.8% to 16.1%, and pleural effusion from 19.4% to 6.5%. Conclusions We established a new assessment scheme for grading disease severity in PTB by specifically considering five radiographic manifestations which were differently associated with the BMI and sputum smear positivity, changed to a different extent after 6 months of treatment and exhibited an excellent agreement between radiologists. Our results suggest that this reading scheme might contribute to the estimation of disease severity with respect to differences in disease pathology. Further studies are needed to determine a correlation with short and long‐term pulmonary function impairment and whether there would be any benefit in lengthening or modulating therapy based on this CXR severity assessment. 113 Mycobacteria, Tuberculosis and Malaria P 85 T cell differentiation capacity in offspring from S. mansoni infected mothers S. Perchermeier1, K. Straubinger1 1
Institut für Mikrobiologie, Immunologie und Hygiene München, Parasitologie, Munich, Germany Infection with the parasitic helminth S. mansoni is characterized by an initial Th1 inflammation, followed by an excessive egg‐induced Th2 response. During the course of infection a long term immunosuppression (Reg phase) is established to protect the host against overwhelming inflammatory responses. This immunosuppression is associated with reduced host responses against parasite antigens as well as bystander antigens, such as allergens. Besides allergy preventing effects within the infected host, there is recent evidence that schistosomiasis during pregnancy influences the offspring´s allergic responses. Previous findings of our group showed that allergic airway inflammation (AAI) in adult offspring from schistosome‐infected mothers is strongly suppressed when pregnancy was initiated during the Reg phase of infection. Thus, we investigated the T cell differentiation capacity in these offspring in more detail. Interestingly, we observed that CD4+CD62L+ naïve T cells from offspring of S. mansoni infected mothers have a strong capacity to differentiate into Th1 cells in vitro, whereas their ability to differentiate into Th2 cells is impaired in comparison to naïve T cells from offspring of uninfected mothers. Epigenetic changes do not seem to be responsible for these findings since no differences in histone acetylation patterns within cytokine promoter regions (IFNγ, IL‐4, IL‐5, IL‐17, FoxP3, RORγt) of naïve T cells were observed. Interestingly, CD4+ T cells from offspring of schistosome infected mothers spontaneously produced IL‐4 in vitro, pointing towards a memory T cell population potentially arisen during prenatal or early postnatal period. Currently, we are investigating the role of memory T cells in more detail with regards to S. mansoni infection in offspring from infected mothers. Here, we specifically focus on the Schistosoma egg antigen (SEA) specific T cell memory pool and its impact on parasite load and immunopathology. Our studies will help to understand the effects of the maternal immune status during pregnancy on T cell differentiation and function during antigen response in the progeny. 114 Mycobacteria, Tuberculosis and Malaria P 86 Eosinophila among patients presenting to a travel clinic H. J. F. Salzer1,2, T. Rolling2, C. D. Vinnemeier2, S. Schmiedel2, J. P. Cramer2 1
2
Forschungszentrum Borstel, Klinische Infektiologie, Borstel, Germany Universitätsklinikum Hamburg‐Eppendorf, I Medizinische Klinik und Poliklinik, Hamburg, Germany Introduction Eosinophilia is an immunologically mediated response to a wide variety of medical conditions and often a challenging task for physicians. Differential diagnoses include allergic diseases, medication use, malignant diseases, endocrine conditions and infectious diseases. New‐onset of eosinophilia among returning travelers is especially seen in helminth infections, particularly during tissue‐invasive disease. Material and Methods Our retrospective study comprised data of 8.354 patients presenting to the Bernhard Nocht Clinic at the University Medical Center Hamburg‐Eppendorf from September 2007 to May 2014. In this period every patient received a standardized questionnaire on travel‐related illness and trip information. Eosinophilia was defined as total eosinophil count ≥ 500 /μL. We excluded patients with a total eosinophil count <500 /μL and failure to complete the questionnaire. Results A total of 6.622 patients were analyzed (1.732 from 8.354 patients were excluded due to incomplete data). In 249 questionnaires eosinophilia was marked. 94 of 255 patients were excluded due to relative eosinophilia (<500/µl). 2.4% (155/ 6.622 patients) showed an absolute eosinophil count ≥ 500 /μL. 60% (95/155 patients) were male and 40% (60/155 patients) female with a median age of 33 years (range 5 ‐ 77 years). 94% (145/155 patients) of patients included presented to the outpatient clinic and 6% (10/155 patients) were inpatients. Most patients presenting with eosinophilia were returning travelers with 82% (121/161 patients) with a medium duration of traveling of 31 days (range 2 to 731 days) followed by 13% immigrants (20/155 patients), 4% tourists visiting Germany (6/155 patients), and 1% expatriates (2/155 patients). An infectious cause could be identified in 57% (89/155) with the most common diagnosis being schistosomiasis in 19% (29/155). No definite cause could be identified in 14% (22/155), while atopic disese and asthma accounted for 10% (16/155). Other reasons for eosinophilia were less common. Patients who were diagnosed with an infectious disease had slightly higher eosinophil counts than those without (median 800 vs 700, p=0.035), but did not differ regarding IgE levels, age or sex. Conclusion An absolute eosinophilia is found in approximately two percent of patients presenting to a travel clinic. While parasitic diseases can explain a large part, their detection can be elaborate. Higher eosinophil counts may point to an infectious cause of eosinophilia. 115 Mycobacteria, Tuberculosis and Malaria P 87 Chronic Q fever endocarditis in a patient with recurrent aortic valve prosthesis P. Vollmar1, B. Thoma1, M. Walter1, U. Reischl2, A. Hiergeist2, S. Feihl3, S. Bleiziffer4, K. Specht5, C. Kahlhofer1 D. Frangoulidis1 1
Bundeswehr Institute of Microbiology, Munich, Germany Institute of Medical Microbiology and Hygiene, Universitätsklinikum Regensburg, Regensburg, Germany 3
Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany 4
Department of Cardiovascular Surgery, German Heart Center Munich, Technische Universität München, Munich, Germany 5
Institute of Pathology, Technische Universität München, Munich, Germany 2
Endocarditis is the most common manifestation of chronic Q fever that occurs in up to 5% of patients with acute infection and valvular heart disease and is caused by the strict intracellular bacterium Coxiella burnetii. Untreated, Q fever endocarditis is fatal, but sufficient therapy of this threatening manifestation is challenging and requires a prolonged combined antimicrobial regime. Herein we report a case of a chronic Q fever endocarditis in a 78‐year‐old woman, which presumably has remained undetected for a long time leading to a recurrent aortic valve and onetime mitral valve prosthesis. The patient was admitted to hospital in December 2014 because of both high grade mitral valve insufficiency and degeneration of a biological aortic valve prosthesis, which had been implanted 15 years previously due to an endocarditis of unknown origin. Surgical valve replacement of both insufficient valves was performed four days after admission. A 16s rDNA sequencing performed from the aortic valve prosthesis revealed Coxiella burnetii, which was in accordance with histopathological findings, showing infiltrations of inflammatory cells in terms of an ongoing endocarditis. Serological testing by means of an indirect immunofluorescence assay showed signs of a chronic Q fever with highly elevated phase I and phase II IgG and IgA antibody titers. Antibiotic treatment with doxycycline 100 mg bid plus hydroxychloroquine 200 mg tid was immediately started for at least 18 months. The treatment regime has been so far well tolerated by the patient except a mild phototoxic skin reaction and all transthoracic echocardiograms controls has been negative so far. A 14 marker Multi‐Locus‐VNTR‐Analysis (MLVA) was performed and identified a genotype seen in 2008 and 2011 in cattle samples from Bavaria, Germany. This observation corresponds well to the patients place of residence located in Bavaria too. The presented case could be attributed to a long time undiagnosed chronic Q fever, leading to recurrent valve failures and highlights that Coxiella burnetii has to be taken in consideration in every case of apparently culture negative endocarditis, according to the Dukes criteria. The therapeutic regime, dual administration of doxycycline and hydroxychloroquine, itself is challenging but essential for patient’s outcome and has to be performed for at least 18 months. Furthermore Q fever serology should be performed when having routine valve surgery. 116 Mycobacteria, Tuberculosis and Malaria P 88 Importance of whole Genome sequencing techniques in Q fever diagnostics D. Frangoulidis1, U. Reischl2, C. Kahlhofer1, M. Walter1 1
2
Bundeswehr Institute of Microbiology, Munich, Germany Institute of Medical Microbiology and Hygiene, Universitätsklinikum Regensburg, Regensburg, Germany The whole genome sequencing of bacteria recently improved rapidly when using so called Next Generation Sequencing techniques. The zoonotic disease Q fever, caused by the strict intracellular Gram‐negative bacterium Coxiella burnetii shows to main clinical entities. An acute course, which is often self limited without further treatment and the chronic disease, resulting from up to 2% of the acute form showing an endocarditis with poor prognosis. Due to its intracellular life cycle C. burnetii diagnostics relies on serology. Isolation of the bacterium is extremely difficult and elaborate and determination of antibiotic resistence pattern is not established. Hence the application of molecular biological methods have had the power to overcome several of these problems. PCR has proven its diagnostic benefits in the very early stage of acute Q fever, whereas it is also a very important tool in culture‐negative endokarditis. Another obstacle is the very low amount of specific DNA in clinical samples which hampers the genotypical analysis. In our recent studies we could show that the application of whole genome amplification techniques, like Multiple displacement amplification (MDA), could enhance the diagnostic abilities. Beside a clear identification of Coxiella house‐keeping genes also the study of the molecular epidemiology of this pathogens was possible. Specific genotypes were identified, matching with known regional genomic variants. To allow a more deeper view in the genome for identification of antibiotic resistance genes and other virulence associated genes we reconstruct the complete genome sequence of Coxiella burnetii from a clinical sample originating from a cardiac valve. This method demonstrates for the first time the ability of modern molecularbiological methods in generating a complete C. burnetii genome from a clinical sample without previous cultural isolation of the bacterium. Our combination of new sequencing methods and bioinformatical analysis tools makes genomic analysis of complex microbiological materials more suitable in the future. 117 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 89 Gender differences in outpatient antibiotic prescribing: A systematic review and meta‐
analysis W. Schröder1,2, H. Sommer3,4, B. P. Gladstone1,2, F. Foschi1,2, J. Hellman5, B. Evengard6, E. Tacconelli1,2 1
Universitätsklinikum Tübingen, Tübingen, Germany German Center for Infection Research (DZIF), partner site Tübingen, Tübingen, Germany 3
Cochrane Germany, Medical Center, Freiburg, Germany 4
Albert‐Ludwigs‐Universität Freiburg, Freiburg, Germany 5
The Public Health Agency of Sweden, Solna, Sweden 6
Umea University, Umea, Sweden 2
Objectives The overall drug prescribing in community shows a substantially difference between males and females that could not only be explained by the incidence of diseases between gender. Of even more concern, individuals receiving antibiotics in primary care are at increased risk of developing colonization and/ or infection due to antimicrobial‐resistant bacteria. Our objective was to systematically review the literature and, where appropriate, meta‐analyse studies investigating prescriptions according to gender in the outpatients setting. Methods MEDLINE and PubMed databases and reference lists of retrieved articles were searched to identify studies analyzing antibiotic prescriptions in primary care published between 1976 and 2013. Experts were contacted for unpublished studies and data. The outcome of interest was the incidence of antimicrobial prescription measured as defined daily dosages (DDD)/ thousand inhabitants (TI) per day as well as prevalence measured as prevalence rate per 1000 inhabitants, stratified by gender, age and antibiotic classes. Two authors independently assessed eligibility of studies and extracted data. Summary statistics were incidence rate ratio (IRR) comparing DDD/TI in males and females. Random effects estimates of the IRR incidence were used. Results Overall, 576 studies were reviewed. 11 studies, including a total of 44,333,839 individuals from nine countries (New Zealand, Spain, Sweden, Belgium, Italy, Israel, Denmark, Germany and Great‐Britain) met the inclusion criteria. The studies were prospective national (7) or regional (4) surveillance from community pharmacy or national health care system. Two databases were unpublished. The risk of receiving an antibiotic prescription was significantly higher in females in respect to men with IRR of 1.25 ± 0.193 and prevalence rate ratio of 1.27 ± 0.122. Specifically, in the age groups 16‐34 (IRR 1.36 ± 0.11) and 35‐54 (IRR 1.40 ± 0.035) and for the antibiotics cephalosporins (IRR 1.44 ± 0.298) and macrolides (IRR 1.32 ± 0.151), which are widely used for respiratory tract infections, major gender differences were observed. Conclusions This meta‐analysis shows that women in the age group 16‐54 receive, in primary care, a significant higher number of prescriptions of penicillin, cephalosporins, and macrolides than men. Current evidence on infectious diseases incidence by gender cannot fully explain such a difference. Since the alarming increase in antibiotic resistance in the community, this result is fundamental for researchers and policy decision‐makers in designing antimicrobial stewardship programs. Prospective studies should be performed to analyze the major determinants for the unequal treatment and their outcomes. 118 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 90 Antibiotic consumption in hospitals: A new system for collection, automated calculation and reporting in Germany B. Schweickert1, M. Feig1, M. Behnke2, L. A. Pena Diaz2, S. Kaersten1, H. Claus3, P. Gastmeier2, M. Abu Sin1 T. Eckmanns1 1
Robert Koch‐Institut, Abt. 3, Fachgebiet für nosokomiale Infektionen und Surveillance von Antibiotikaresistenz und Antibiotikaverbrauch, Berlin, Germany 2
Institut für Hygiene und Umweltmedizin, Universitätsmedizin Charité, Berlin, Germany 3
Robert Koch‐Institut, Abt. 3, FG Datenmanagement, Berlin, Germany Introduction Since 2011, German hospitals are legally obliged to monitor antibiotic consumption. The national public health institute (Robert Koch Institut), in cooperation with the National Reference Center for the Surveillance of Nosocomial Infections, built up a system for data collection, calculation and reporting. Aims The aims of the project are to support the hospitals in implementation and conduct of antibiotic consumption surveillance and local antibiotic stewardship efforts and to provide benchmark data. Methods For standardized data provision and efficient data processing an electronic system has been developed. The already existing web‐based data portal (“webKess”), which serves for the collection of data in the German Hospital Infection Surveillance System (KISS) has been extended in order to allow for the entry of antibiotic consumption data and the consecutive transfer to the Robert Koch Institute. This construct also paves the way for future crosslinking of data from the different surveillance systems. Methodological basis is the WHO‐ATC (Anatomical Therapeutic Chemical/DDD (Defined Daily Dose) method. Target measure is the quantity of DDD in relation to 100 bed days and admissions, respectively, calculated for the different medical specialities and ward types. Results The data flow can be divided into three major steps, which are schematically outlined in Figure 1. 1. Upload and transfer: Data upload takes place via a web‐
based tool, which allows manual data entry as well as the bulk import of whole data sets. Three different data files are required: One data set containing data on hospital structure, a second and third dataset cont aining data on antibiotic consumption and hospital activity. Structurally correct datasets are transferred to the Robert Koch Institute for further data processing. 2. Data analysis: After undergoing validity checks concerning content and technical aspects, the data are merged in order to calculate the quantities of antibiotic consumption standardized by bed days and admissions, respectively. 3. Feedback: Reports can be retrieved by password‐protected access via an interactive database, which allows a specification and tailoring of the request (e.g. concerning ward type, speciality, time period) according to the needs and preferences of the user. The system offers different report types supporting various forms of interpretation: a basic report containing data from successive time periods in order to allow the analysis of trends, a ranking list and a report comparing data of the individual hospital and aggregated data of reference hospitals. Figure 1 Conclusion An electronic system for data upload, automated processing and reporting has been built up providing a suitable instrument for local antibiotic stewardship activities as well as the technical capacity for the establishment of a national data base. 119 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 91 Management und Prognose der Staphylococcus aureus‐Bakteriämie am Universitätsklinikum Göttingen M. H. Schulze1, S. Öztürk Mert1, A. Dudakova1, U. Groß1 1
Universitätsmedizin Göttingen, Institut für Medizinische Mikrobiologie, Göttingen, Germany Fragestellung Die Staphylococcus aureus (S. aureus)‐Bakteriämie (SAB) weist trotz Fortschritte in Diagnostik und Therapie nach wie vor eine hohe Mortalität auf. In Abhängigkeit vom Patientenklientel bewegt sich diese zwischen 15 und 50 % (1). Im Rahmen der Umsetzung von Antibiotic Stewardship (ABS) an der Universitätsmedizin Göttingen wurde 2013 ein Pilotprojekt initiiert, durch das alle Patienten mit SAB ein Konsil durch den klinischen Infektiologen des ABS‐
Teams erhalten. Dieses soll den betreuenden Kliniker unterstützen, das Management und die Prognose der betroffenen Patienten zu verbessern. Die erhaltenen Daten werden mit einem 12‐monatigen Zeitraum vor Beginn des Pilotprojektes verglichen. Methoden Das Universitätsklinikum Göttingen hat eine Kapazität von 1.456 Betten. Jährlich werden zirka 54.000 Patienten stationär aufgenommen. Pro Jahr treten im Durchschnitt 120 Fälle einer SAB im gesamten Klinikum auf. Das ABS‐Konsil findet in der Regel zwischen 48 und 72 Stunden nach Abnahme der ersten Blutkultur mit Nachweis von S. aureus statt. Anhand eines standardisierten Konsil‐Scheines wird Patienten‐individuell die weitere Therapie, Therapiedauer und Diagnostik festgelegt. Ergebnisse Von den prospektiv seit 2013 in die Datenbank aufgenommenen Patienten sind bisher 38 vollständig ausgewertet. Das mediane Alter beträgt 70 Jahre; Range 53‐89; Mean 70,6 Jahre. Von den 38 Patienten sind 9 (23,7%) aufgrund der SAB verstorben (Überleben: Median 7 Tage; Range 1‐26 Tage; Mean 8,3 Tage). Weitere 9 (23,7%) Patienten sind im Verlauf aufgrund einer anderen nicht‐infektiösen Erkrankung verstorben (Überleben: Median 25 Tage; Range 7‐351 Tage; Mean 63,6 Tage). Schlussfolgerungen Die SAB ist trotz optimalem Managements mit einer hohen Mortalität innerhalb der ersten 30 Tage assoziiert. Ein weiterer großer Teil der Patienten mit erfolgreich behandelter SAB verstirbt innerhalb eines Jahres aufgrund nicht‐infektiöser Ursachen. Der Vergleich der prospektiven mit den retrospektiven Daten ist ausstehend. Literatur 1. van Hal SJ, Jensen SO, Vaska VL, Espedido BA, Paterson DL, Gosbell IB. Predictors of mortality in Staphylococcus aureus Bacteremia. Clin Microbiol Rev. 2012;25(2):362‐86. 120 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 92 Evaluation of the Necessity of an Antibiotic Stewardship Programme at a Paediatric Tertiary Care Centre K. Kreitmeyr1, U. von Both1, A. Pecar2, J. Hübner1, M. Andraschko2 1
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Dr. von Haunersches Kinderklinikum, LMU, Infektiologie, Munich, Germany Klinikum der Universität München , Apotheke, Arzneimittelinformation, Munich, Germany Introduction Previous data suggest that up to 50 % of hospitalized patients receive antibiotic therapy and 20‐50 % of all antibiotics prescribed in acute care hospitals are either unnecessary or inappropriate. This causes severe side effects, avoidable healthcare expenditures and, in addition, is key in driving increasing rates of antibiotic resistance. In order to ensure continuity of successful treatment of infectious diseases (ID), the WHO released a global action plan demanding universal implementation of Antibiotic Stewardship (ABS) programmes to avoid inappropriate use of antibiotics. Objectives We analysed the trend of overall antibiotic usage in four paediatric wards of the Dr. von Hauner Children's Hospital during 2011 and 2014 and evaluated the accuracy of choice and dosing of antibiotics at bedside in a prospective 4‐month period. Particular focus was laid on the extent of precise therapeutic drug monitoring (TDM) for antibiotics with narrow therapeutic ranges. Patients and Methods Analysis of antibiotic usage of the four selected wards was based on data obtained from our hospital pharmacy. To evaluate individual treatments, we documented patient characteristics (age, body weight, etc.), clinical and laboratory findings as well as specific therapeutic parameters (substance, dosage, route of administration, presence of TDM results, etc.) for all patients receiving any antibiotic between 01.09.2014 and 31.12.2014. Results Our analysis demonstrates that antibiotic‐related costs could be cut by >50 % by implementation of prescribing restrictions and prospective audit with feedback as first ABS‐strategies in 2012 and showed that this effect was sustainable throughout 2014 (Huebner et al. Klin Padiatr 2013; 225(04): 223‐229). For a patient‐related evaluation we collected data of 268 individuals during the 4‐month bedside study period. Out of 528 prescribed antibiotics, the dose of only 76.4 % was within a rather wide range of ±30 % of national dosing recommendations. According to this standard, 17.5 % of administered antibiotics were underdosed and 6.1 % overdosed. We observed that TDM was performed in only 8 out of 21 (38 %) patients treated with Vancomycin, an antibiotic where TDM is strongly recommended due to its narrow therapeutic range. In these monitored patients, target range serum levels were only achieved in 28.8 % of treatment time. Conclusion Our data demonstrate that there is still a great need for improvement of antibiotic therapies in our tertiary care centre. To avoid incorrect antibiotic dosing and to attain correct monitoring of aminoglycosides are currently amongst our main objectives. Implementation of precise dosage recommendations, guidelines on correct TDM and interdisciplinary discussions during regular educational paediatric ID ward rounds are main ABS strategies and will most certainly prove as effective as previously shown in adults. 121 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 93 Strategies to reduce inadequate antibiotic prescriptions for upper respiratory tract infections in outpatient care J. Zweigner1,2, M. Wiese‐Posselt2, A. M. Rohde2, P. Gastmeier2 1
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Uniklinik Köln, Zentrale Krankenhaushygiene, Cologne, Germany Charité‐ Universitätsmedizin Berlin, Institut für Hygiene und Umweltmedizin, Berlin, Germany Introduction In Germany, 85 % of all antibiotics are prescribed in the outpatient care, mostly for the treatment of upper respiratory tract infections (URTI) although these are caused predominantly by viruses. Patients’ knowledge and expectations may influence these prescriptions of antibiotics. However, providing information during consultation is challenging, thus patient information material could facilitate this. Objectives Aim of this study was to provide patient information leaflets and brochures about the development of bacterial resistance, cause and treatment of URTI in order to facilitate consultations and reduce antibiotic prescriptions. Methods As part of the antibiotic therapy optimisation study (ATHOS) we developed together with Lindgrün GmbH, a detailed patient information brochure, a concise consultation leaflet, an infographic poster and participation poster for general practitioners (GP) and patients. Additionally, an advanced training course for GPs was designed and offered five times between October and November 2014 in Berlin to address the problem of bacterial resistance, inadequate antibiotic prescription for URTI and to introduce the materials. Supported by the Kassenärztlichen Vereinigung Berlin, 2359 GPs were invited to participate in the advanced training course and receive all the materials on request. Results 164 (6.98 %) of the invited GPs attended the advanced training course. 64.2 % of the participants evaluated the course and material. 65.7 % of them considered the content of the training course as relevant for their own practice and 62.1 % believed the material could influence patients knowledge and expectations. 63 patients were asked to evaluated the material. 79 % were pleased with the consultation leaflet and 59 % with the patient brochure. To date, 74 GPs have ordered 4635 patient brochures, 2745 consultation leaflets, 469 infographic posters and 465 participation posters. Conclusion Patient brochures and leaflets about URTI providing information about cause of symptoms, self‐management and treatment were appreciated both by GP and patients. They are promising tools to facilitate consultations, to increase patients’ knowledge and may effectively decrease antibiotic prescriptions for the treatment of URTI. 122 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 94 Spread of fluoroquinolone resistant CTX‐M‐15‐producing Escherichia coli ST410 in humans and animals L. Falgenhauer1, C. Imirzalioglu1, H. Ghosh1, K. Gwozdzinski1, J. Schmiedel1, K. Gentil1, T. Chakraborty1 1
University Hospital Giessen, Institute of Medical Microbiology, Giessen, Germany Multidrug‐resistant Escherichia coli from humans, companion animals, and livestock frequently harbour extended‐spectrum beta‐lactamase genes, thereby impairing the treatment options in case of an infection with bacteria carrying these enzymes. In particular, CTX‐M‐type ESBL enzymes isolates are frequently isolated from humans, companion animals, livestock, wild animals and the environment, raising concern regarding exchange and spread of isolates among these populations. To address this question, we performed detailed molecular epidemiological analysis of ESBL‐producing E. coli in animal and human populations in Germany. Whole genome sequencing was performed for 94 CTX‐M‐15‐producing E. coli isolates from humans (n=47), companion animals (n=26), livestock (n=17), and farm environments (n=4). The sequence type (ST) of these isolates was identified. Phylogeny and single nucleotide polymorphisms (SNPs) of sequenced isolates were assessed. Virulence genes and plasmid properties were identified. 26 STs were detected among the CTX‐M‐15‐producing E. coli isolates. ST410 was the most frequent ST found and was isolated from humans (n=9), companion animals (n=4), livestock (n=8), and the farm environment (n=3). Within the ST410 isolates, we identified five clades (A‐E). Isolates of clade B were present in all four populations and their core genomes differed by less than 75 SNPs from each other. In addition, isolates of clade B and C were clonally marked by chromosomal insertion of blaCTX‐M‐15 genes either in the rhsE locus (clade B) or in a defective lambdoid bacteriophage (clade C). Our data provides strong evidence for clonal dissemination of CTX‐M‐15‐producing E. coli ST410 between human and animal populations. 123 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 95 Clinical and microbiological factors in colonization with extended spectrum beta‐lactamase producing Enterobacteriaceae in high‐risk patients ‐ preliminary results from the EMERGE cohort L. M. Biehl1,2,3, B. Stecher4,5,6, D. Garzetti4,5,6, M. Koeppel4,5,6, C. Imirzalioglu7,8, C. Belmar Campos3 H. Rohde3,9,10, M. J. G. Vehreschild1,2,10 1
University Hospital of Cologne, Department I of Internal Medicine, Cologne, Germany German Centre for Infection Research (DZIF), Partner site Bonn‐Cologne, Germany 3
University Medical Center Hamburg‐Eppendorf, Institute for Medical Microbiology, Virology and Hygiene, Hamburg, Germany 4
Ludwig‐Maximilians‐University, Max‐von‐Pettenkofer Institute, Munich, Germany 5
German Centre for Infection Research (DZIF), Partner site Munich, Germany 6
DZIF Center for Gastrointestinal Microbiome Research (CEGIMIR), Munich, Germany 7
Justus‐Liebig University, Institute of Medical Microbiology, Giessen, Germany 8
German Centre for Infection Research (DZIF), Partner site Giessen‐Marburg‐Langen, Germany 9
German Centre for Infection Research (DZIF), Partner site Hamburg‐Borstel, Germany 10
† Authors contributed equally, Germany 2
Background In immunocompromised patients, the influence of clinical and microbiological factors, particularly the gut microbiota on the course of colonization with multi‐resistant bacteria like extended spectrum ß‐lactamase‐
producing Enterobacteriaceae (ESBL‐E) and vancomycin‐resistant Enterococci (VRE) is largely unknown. Recent studies indicate that cytotoxic therapy, antibiotic treatment and presence of inflammation lead to a shift in the composition of gut microbiota and potential expansion of multiresistant organisms, thus facilitating bloodstream infections and horizontal gene transfer (HGT). To further elucidate the underlying processes, a longitudinal study assessing clinical patient data, colonization with ESBL‐E and VRE, presence of plasmid transfer, as well as changes in the gut microbiome, was set up. Methods Patients were eligible for inclusion in the longitudinal cohort if they were either scheduled for intensive cytotoxic treatment for a minimum of 3 weeks or if they were already found to be colonized with an ESBL‐E or VRE as oncological inpatients. Rectal swabs were obtained weekly during inpatient treatment. An aliquot of the swab medium was frozen at ‐80°C for microbiome analysis. Detection of ESBL‐E and VRE was performed using enrichment broth and selective agars. Suspected isolates were differentiated using MALDI‐TOF and subjected to resistance testing. Follow‐up isolates were compared by pulsed‐field gel electrophoresis (PFGE). Presence of resistance genes will be analyzed using a micro array. A subset of isolates will be analyzed for clonality, HGT and virulence genes using whole genome sequencing (WGS). Furthermore, genomic DNA will be extracted from all retained fecal samples and subjected to 16S rRNA gene sequencing of the V3‐V4 regions. Results From July 2014 to March 2015, 42 patients were included in the study. Overall, 421 fecal swabs were collected (range 1‐30 per patient). 17 patients were found to be colonized with ESBL‐E and 10 with VRE. 3 ESBL‐E colonizations and 5 VRE colonizations emerged during the observational period. PFGE revealed the presence of distinct ESBL‐E genotypes of the same species in 5 cases (all E. coli). Presence of different species of ESBL‐E was observed in 3 cases. There were 3 consecutive bloodstream infections due to isolates clonally identical to colonizing ESBL‐E. The protocol for DNA extraction and 16S rRNA sequencing has been validated using test samples showing reproducible and reliable results for rectal swabs in these settings. Conclusion In this longitudinal cohort of immunocompromised patients different courses of colonization including subsequent bloodstream infection and presence of diverse ESBL‐E could be observed allowing for further investigation of associated microbiome changes. In some cases there is a high suspicion of the occurrence of HGT, which will be analyzed further using WGS. 124 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 96 Sulfide production and polysulfide detoxification contributes to antibiotic resistance in Staphylococcus aureus N. Ritzmann1, D. Nusser1, J. Weikum1, F. Nadeu Prat1, A. Siebert1, H.‐ G. Sahl 1, F. Grein1 1
Institut für Pharmazeutische Mikrobiologie, Bonn, Germany Reactive oxygen species (ROS) are not only one of the major weapons in the hosts’ armory against pathogenic bacteria, but also contribute to the bactericidal effect of antibiotics as they cause fatal destruction due to their high reactivity with many cellular components such as proteins, lipids or DNA. Therefore, staphylococcal defense mechanisms against oxidative stress may represent an alternative target for novel antiinfectives. Recently it was found that bacteria can protect themselves significantly from ROS and as a consequence from antibiotics of various classes by the production of sulfide (Shatalin et al. 2011). The proposed mechanisms are mainly the depletion of cellular iron and cysteine, both of which contribute to the generation of extremely harmful hydrogen peroxide radicals. Additional mechanisms are the activation of ROS detoxifying enzymes and the direct neutralization of hydrogen peroxide. We are interested in the role of sulfide production as part of antibiotic resistance in Staphylococcus aureus. We show here that externally added sulfide fully protects S. aureus in particular from killing by aminoglycoside antibiotics. Furthermore inhibition of the sulfide synthesizing enzymes cystathionine ß‐synthase (CBS) and cystathionine γ‐lyase (CSE) increased aminoglycoside susceptibility. Studies are ongoing to reveal if the sulfide‐mediated protection relies on the inhibition of the energy‐dependent drug uptake mechanism in addition to the mechanisms proposed by Shatalin et al. (2011). The protective sulfide, however, was found to be rapidly oxidized to polysulfides which did not protect from aminoglycoside mediated killing and which were notably found to be toxic at higher concentrations. An enzyme cluster composed of a sulfur transferase (ST), a sulfur dioxygenase (SDO) and a sulfide:quinone oxidoreductase (SQR) was identified to be responsible for detoxification of polysulfides. This cluster is partially duplicated in methicillin‐resistant Staphylococcus aureus (MRSA) strains and was previously proposed to be responsible for sulfide oxidation (Luebke et al. 2014). The two enzyme clusters responsible for production of sulfide (CBS/CSE) on the one hand and detoxification of polysulfides (ST/SDO/SQR) on the other hand are thought to be an effective system for mediating antibiotic non‐susceptibility in S. aureus and are therefore under further investigation as anti‐staphylococcal targets. Shatalin et al. (2011) H2S: a universal defense against antibiotics in bacteria. Science 334, 986‐990 Luebke et al (2014) The CsoR‐like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus. Mol. Microbiol. 94, 1343‐1360 125 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 97 DZIF TTU 08.701 Clinical Research Unit, Universitätsklinikum Tübingen W. Schröder1,2, F. Foschi1,2, K. Spohn1,2, S. Armean1,2, E. Tacconelli1,2 1
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Universitätsklinikum Tübingen, Tübingen, Germany German Center for Infection Research (DZIF), partner site Tübingen, Tübingen, Germany The foundation of Clinical Research Unit (CRU) was meant to improve translational research in the field of health‐care associated infections involving microbiological, clinical and basic science. The CRU allows the conduction of clinical trials related to Infectious Diseases and offers an excellent opportunity for exchange between scientists and clinicians. The CRU platform, currently employing two scientists and one research nurse, runs studies focused on the clinical and epidemiological aspects of healthcare associated infections (HAIs). In detail, the CRU provides support, samples and patient data for DZIF clinical trials (8.801 and 6.803) running through the University Hospital Tübingen. For the SPECTRUM study (6.803) 434 patients were included according to study criteria and 143 patients could provide two completed samples. Related data and faecal samples were stored. For clinical study ATHOS (8.801) Tübingen functions as a control hospital. Two prevalence phases with a screening of patients for third generation cephalosporin resistance Enterobacteriaceae (3GCREB) and Vancomycin resistant Enterococci (VRE) in 2014 (407 Patients, prevalence rate of 9%, no VRE) and the same amount in 2015 (screening ongoing) were performed. In parallel, incidence surveillance of VRE, 3GCREB and CDAD as well as antibiotic consumption analysis is ongoing and under analysis. One new clinical study (“Pharmacokinetic and pharmacodynamic evaluation of Vancomycin during intermittent and continuous infusion”) was in the process of protocol set up. Two meta‐analyses (“Meta‐analysis on the effect of antimicrobial stewardship on the colonisation and infection due to antimicrobial resistant strains in hospitalised patients” and “Gender differences in outpatient antibiotic prescribing: A systematic review and meta‐analysis”) were performed. One further research project (“Modelling the effect of selective digestive tract decontamination (SDD) against ESBL‐producing Enterobacteriaceae on the rate of bloodstraem inefctions (BSI) among neutropenic patients”) was finalised. Education is a major focus for the CRU. Two professional education courses (“12. Symposium Infektionsmedizin in Tübingen” and “How to design and perform your clinical studies in Infectious Diseases and Clinical Microbiology”) were organised and held in Tübingen in order to educate future Infectiologists in Germany. The CRU is also involved in different collaborative European projects, namely DRIVE‐AB‐2014 (“Driving Re‐
investment in R&D and responsible antibiotic use“; IMI 2014) and EPI‐NET in COMBACTE‐MAGNET (IMI topic 6A ND4BB, Increasing capabilities in epidemiology). The CRU reached a prominent position in the research on HAIs at international level and will further progress in developing new projects and educational tools in order to reduce the burden related to HAIs. 126 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 98 Characterization of outer membrane vesicles from Gram‐negative bacteria and the role of hemolysin F in vesicles formation. K. Gwozdzinski1, L. Falgenhauer1, M. A. Mraheil1, M. A. Marahiel2, C. Imirzalioglu1, T. Chakraborty1 1
University Hospital Giessen, Institute of Medical Microbiology, Giessen, Germany Philipp University of Marburg, Department of Chemistry, Biochemistry and LOEWE‐Center for Synthetic Microbiology, Marburg, Germany 2
Outer membrane vesicles (OMVs) are spherical, bilayered phospholipids secreted ubiquitously from all Gram‐
negative and some Gram‐positive bacteria investigated to date. The contributions of OMVs to biological processes are diverse and include the release of virulence factors, signaling between bacterial and eukaryotic cells, DNA transfer, antibacterial activity and immunomodulation of the host. This study focuses on the characterization of OMVs isolated from various Gram‐negative bacteria. OMVs were isolated from bacterial cultures of E. coli, Enterobacter 247, S. marcescens, A. baumannii and C. freundii by differential centrifugation and filtration. OMV purity was confirmed by transmission electron microscopy and bacterial culture. OMV fractions were resolved by SDS‐PAGE and subsequently stained with CCB. Proteins were identified by mass spectrometry and protein‐protein interactions were visualized by Cytoscape software based on experimentally determined databases. Protective immune responses induced by OMVs were determined by infecting the moth larvae Galleria mellonella. Larvae were treated with different concentration of native and heat‐treated OMVs 24 hours prior to bacterial challenge and the survival rates were monitored for 7 days. Hemolysin F (hlyF) was recombinantly expressed in E. coli DH10β and OMV production was compared with hlyF‐negative E. coli isolates. OMVs derived from tested gram‐negative bacteria are spherical shaped, mono‐ and bilayered structures ranging from 20 to 250 nm in diameter. CBB stained SDS‐PAGE of whole‐cell lysates and outer membrane vesicles demonstrated differences in the protein content between these two fractions, suggesting that a specific protein sorting mechanisms exist when OMVs are released. Cytoplasmic, abundant outer membrane and periplasmic proteins were found in OMVs by mass spectrometry. In addition, protective immune responses induced by OMVs was examined. Priming G. mellonella with OMVs potently conferred protection against bacteria‐induced lethality.Furthermore, the contribution of HlyF to hyper‐vesiculation was assessed. An in‐silico analysis identified a NAD‐dependent epimerase/dehydratase activity in hlyF. Hemolysin F contributed to increased OMVs production as shown by TEM, β‐lactamase activity and protein concentration. Collectively, the results described above illustrate that all investigated Gram‐negative bacteria constitutively shed OMVs to surrounding milieu and they are involved in various biological functions. Future research will focus on the mechanism underlying vesicle formation in the presence of hlyF and the role of OMVs in bacterial pathogenesis. 127 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 99 Molecular epidemiology of an outbreak of multi‐resistant Acinetobacter baumannii reveals dynamic changes involving antibiotic resistance Y. Yao1,2, C. Imirzalioglu 1,2, H. Ghosh1,2, R. Auer1,2, L. Falgenhauer1,2, B. Bunk3,4, C. Spröer3,4 G. Hemmrich‐Stanisak5, P. G. Higgins6,7, K. Prior8,9, H. Fickenscher6, H. Seifert6,7, D. Harmsen8,9, A. Goesmann2,7 J. Overmann3,4, A. Franke5, T. Chakraborty1,2 1
Institute for Medical Microbiology, Justus‐Liebig‐University Giessen, Giessen, Germany German Center for Infection Research (DZIF), Partner Site Giessen‐Marburg‐Langen, Campus Giessen, Giessen, Germany 3
Leibniz Institute DSMZ‐German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany 4
German Center for Infection Research (DZIF), Partner Site Hannover‐Braunschweig, Braunschweig, Germany 5
Institute of Clinical Molecular Biology, Christian‐Albrechts‐University Kiel, Kiel, Germany 6
Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany 7
German Center for Infection Research (DZIF), Partner Site Cologne‐Bonn, Cologne, Germany 8
Department of Periodontology, University Hospital Münster, Münster, Germany 9
German Center for Infection Research (DZIF), Associated Partner of Partner Site Borstel, Borstel , Germany 10
Institute for Infection Medicine, UKSH, Kiel, Germany 11
Bioinformatics Computational Biology, Justus‐Liebig‐University Giessen, Giessen, Germany 2
Prevalence of nosocomial infections and outbreaks with multi‐drug‐resistant (MDR) Acinetobacter baumannii has increased in recent years. A large outbreak at an university hospital in Germany caused by MDR A. baumannii involving over 35 patients and 14 deaths between December 2014 and February 2015 was analysed by whole genome sequencing (WGS) for genetic relatedness and transmission dynamics. Genomes of 45 bacterial isolates from patients and the hospital environment were sequenced using Illumina Sequencing Technology (NextSeq) and a subset was examined by single‐molecule real‐time (SMRT) sequencing (Pacific Biosciences). Genome Assembly was performed by using various Assemblers (CLC Genomics Workbench, Spades and HGAP3). Genomic phylogeny, Core Genome SNPs and comparative genomic analysis were carried out using GenDB, Edgar, Mauve, CLC and Harvest‐Parsnp. Phylogenetic analysis revealed a clonal outbreak with uniform genetic antibiotic resistance patterns. The outbreak clones were members of the international clone IC2 (ST‐2) and Oxford sequence type ST‐195. All strains harboured an 8 kb plasmid similar to pAC12. We used single nucleotide polymorphisms (SNPs) to track transmission dynamics and detected distinct sub‐
clones suggesting that variant isolates were already present in the hospital environment prior to their detection in the index case. Indeed comparative genomics indicated that outbreak isolates were closely related to independent isolates previously detected in three separate outbreaks from Northern and Western Germany. Comparative analysis of closed genomes of four representative isolates from the current and previous outbreaks demonstrated that isolates were virtually identical. Outbreak‐specific unique genes are largely associated with the presence of distinct prophages present in the respective genomes. We detected dynamic changes in genomic content involving antibiotic resistance to colistin and the loss of a 29 kb genetic island harbouring a gene encoding sulfonamide resistance sul2. WGS is a valuable technique for tracing transmission and origins during an outbreak. Our data suggests that this outbreak involved strains that were probably already indigenous to the hospital environment. 128 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 100 Deficits in the practice of blood culture sampling in Germany ‐ results of a knowledge, attitude and practice study among physicians in Lower Saxony and Bavaria A. Karch1,2, J. Hübner3, A. Duddeck2, H. Raupach‐Rosin2, R. T. Mikolajczyk1,2 1
German Centre for Infection Research, Hannover‐Braunschweig site, Braunschweig, Germany Helmholtz Centre for Infection Research, ESME ‐ Epidemiological and Statistical Methods Research Group, Braunschweig, Germany 3
Dr. von Hauner Children's Hospital, Munich, Germany 2
Background Recent studies suggest that blood culture sampling rates as well as blood culture positivity rates in Germany are considerably lower than recommended. This might indicate deficits in both, decision on blood culture ordering and implementation of blood culture collection guidelines. Aim of our study was therefore to assess knowledge, attitudes and practice of physicians in Germany regarding blood culture diagnostics. Methods We conducted a cross‐sectional study among physicians working in inpatient care in the federal states of Lower Saxony and Bavaria. Department heads were contacted and asked for distributing the study invitation in their departments. In total, 293 medical doctors agreed to participate and completed an online questionnaire with 52 questions. Descriptive statistical methods were used to determine knowledge and attitudes regarding blood culture diagnostics. In order to assess the quality of individual blood culture practice according to existent guidelines, a six‐item score was constructed. Using this score, we applied a multivariable linear regression model to identify predictors for good blood culture practice on the individual and institutional level. Results Blood culture sampling was considered an important tool for the diagnosis of bloodstream infections by 95% of the participating physicians. However, only 17% of them decided to collect blood cultures in three constructed scenarios for which blood culture ordering was recommended by present guidelines; almost one out of ten physicians wouldn’t have taken blood cultures in any of the three scenarios. The majority of participants (78%) reported not to adhere to the guideline recommendation that blood culture sampling should include at least two blood culture sets from two different injection sites. According to the multivariable regression analysis, high routine in blood culture sampling, perceived importance of blood culture diagnostics and the existence of institutional standards for blood culture sampling were identified as predictors for good blood culture practice. Conclusion Our study suggests that there are substantial deficits in blood culture ordering and the applications of guidelines for good blood culture practice in Germany. Based on these findings, we suggest that a multimodal intervention concept targeting the individual as well as the institutional level might be suitable for improving blood culture diagnostics in German hospitals. 129 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 101 Emergence of ciprofloxacin resistant Salmonella enterica serovar Typhi in East Africa D. Eibach1, H. Al‐Emran1, D. Dekker1, R. Krumkamp1, F. Marks2, J. May1 1
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Bernhard‐Nocht‐Institut für Tropenmedizin, Hamburg, Germany International Vaccine Institute, Seoul, South Korea Background Salmonella enterica serovar Typhi (S. Typhi) are a predominant cause of bloodstream infections in sub‐Sahara Africa (sSA). Resistance to ciprofloxacin has been increasingly reported worldwide, however data from sSA are limited. This study aims to assess ciprofloxacin susceptibility of S. Typhi isolates from febrile patients in sSA and to determine their resistance mechanisms. Methods From seven study sites (Burkina Faso, Ghana, Guinea‐Bissau, Kenya, Madagascar, Senegal and Tanzania) blood culture samples from patients with a temperature ≥38.0°C were obtained. After biochemical identification antimicrobial sensitivity testing was performed for all Salmonella isolates by disc diffusion test and ciprofloxacin Etest. Salmonella with reduced ciprofloxacin susceptibility were screened for mutations in the gyrA, gyrB, parC, parE and plasmid‐mediated quinolone resistance genes by PCR. Results Out of 13,372 blood cultures, 191 S. Typhi, two S. Paratyphi A and 223 non‐typhoid Salmonella (NTS) were isolated. Multidrug resistant (MDR) S. Typhi were found in Kenya (80%, n=39) and Tanzania (89%, n=8). Reduced susceptibility to ciprofloxacin was present in 11 isolates (22%) from Kenya and revealed a Glu133Gly mutation in the gyrA gene in combination with either another gyrA (Ser83Phe) or gyrB mutation (Ser464Phe). One S. Paratyphi A isolate, non‐susceptible to ciprofloxacin, was foundin Senegal with mutations in the gyrA (Ser83Phe) and parC (Ser57Phe) genes. No mutations were detected in parE and PMQR genes. Conclusion A substantial number of ciprofloxacin and MDR S. Typhi were found in East Africa while S. Typhi isolates from West Africa and Madagascar remain pan‐susceptible. In Kenya, physicians should be aware that empirical antimicrobial therapy with ampicillin, chloramphenicol, TMP‐SMX and ciprofloxacin for S. Typhi infections might not be effective. In West Africa continuous monitoring of antimicrobial susceptibility is required to detect ciprofloxacin resistant isolates in a timely manner and to adjust treatment regimes accordingly. 130 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 102 Incidence of multiresistant organisms and Clostridium difficile infections in 6 German university hospitals A. M. Rohde1, J. Zweigner1,2, M. Wiese‐Posselt1, M. Behnke1, W. Kern3, P. Gastmeier1, H. Seifert2 DZIF‐ATHOS Study Group1,2,3,4,5,6 1
Charité Berlin, Institut für Hygiene und Umweltmedizin, Berlin, Germany University Hospital Cologne, Institue of Medical Microbiology, Immunology and Hygiene, Cologne, Germany 3
University Hospital Freiburg, Departement for Infectiology, Freiburg, Germany 4
University Hospital Tübingen, Departement for Infectiology, Tübingen, Germany 5
University Hospital Klinikum Rechts der Isar, Munich, Germany 6
University Hospital Schleswig‐Hostein, Lübeck, Germany 2
Introduction This report is part of the multicenter study ATHOS (antibiotic therapy optimisation study). ATHOS aims at collecting prevalence and incidence data for nosocomial carriage of multi‐drug resistant organisms and to intervene in the inpatient and outpatient setting. Objectives The aim of this study was to assess the incidence of third generation cephalosporin‐resistant enterobacteria that are resistant against three or four out of four antimicrobial classes (3MRGN, 4MRGN), vancomycin‐
resistant enterococci (VRE) and Clostridium difficile infections (CDI) as baseline data for an antibiotic stewardship intervention in the inpatient setting. Methods From January to September 2014, six German university hospitals monitored their patients for 3GCREB, VRE and CDI occurrence, their number of patients and patient days. 3MRGN, 4MRGN and VRE cases were defined as microbiological finding in a clinical sample and infection as a case with antimicrobial therapy (3MRGN, 4MRGN and VRE). CDI cases were defined as loose stools with microbiological finding of C. difficile toxins. CDI relapses (within 8 weeks after the first finding) were not counted. Findings from the patient’s first three days of stay were classified as community‐acquired and findings from patient day 4 on as hospital‐acquired. The incidence was calculated as cases per 100 patients and the incidence density as cases per 1000 patient days. Patients from paediatrics, psychiatry, dermatology, ear nose and throat medicine and ophthalmology were excluded. Results In a preliminary analysis, 196 383 patients with 1 255 876 patient days were included. Hospital‐acquisition of 3MRGN occurred in 0.26 cases per 100 patients (range: 0.20‐0.38), or respectively in 0.02 cases per 100 patients (4MRGN; range: 0.00‐0.07) and for VRE in 0.12 cases per 100 patients (range: 0.02‐0.25). The incidence density of 3MRGN was 0.40 (cases per 1000 patient days; range: 0.23‐0.52) and 0.27 (infections per 1000 patient days; range: 0.15‐0.41). The incidence density of 4MRGN per 1000 patient days was 0.04 (cases; range: 0.00‐0.09) and 0.02 (infections; range: 0.00‐0.04). The incidence density of VRE per 1000 patient days was 0.19 (cases; range: 0.04‐0.28) and 0.10 (infections; range: 0.02‐0.24). Hospital‐acquisition of CDI occurred 0.31 cases/100 patients (range: 0.15‐0.99). The incidence density of CDI was 0.49 per 1000 patient days (range: 0.22‐1.28). Conclusion In our analysis, we observe a high variability among the six study centres, especially for VRE and CDI. This difference is only partly explainable by the number of community‐acquired cases of multiresistant organisms and CDI and therefore demands further study. 131 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 103 Inzidenz nosokomialer Clostridium difficile‐assoziierter Diarrhoe und Antibiotikaverbrauch: Eine erste Analyse innerhalb der ATHOS‐Inzidenzstudie V. Ihle1, A. Mischnik1, N. Jazmati2, J. J. Vehreschild2, G. Langebartels2, A. Liekweg2, M. Hug1, G. Häcker1 M. Steib‐Bauert1, H. Seifert2, W. Kern1, U. D. ATHOS‐Studiengruppe3 1
Universitätsklinikum Freiburg, Freiburg, Germany Universitätsklinikum Köln, Cologne, Germany 3
DZIF Deutsches Zentrum für Infektionsforschung, Cologne, Germany 2
Einleitung Ein vermehrter Einsatz von Drittgenerations‐Cephalosporinen (3GC) und Fluorchinolonen (FQ) scheint eine hohe Inzidenz von Clostridium difficile‐assoziierter Diarrhoe (CDAD) zu begünstigen. Ziel der DZIF‐unterstützten „Antibiotika‐THerapie‐Optimierungs‐Studie“ (ATHOS) ist die Prüfung, ob mit Änderungen des Einsatzes von 3GC+FQ über Antibiotic Stewardship (ABS)‐Programme die Inzidenzdichte nosokomialer CDAD und nosokomialer Infektionen durch multiresistente gramnegative Bakterien reduzierbar ist. Methoden In mehreren Universitätskliniken werden seit 2014 prospektiv CDAD‐Fälle auf ATHOS‐Teilnehmerstationen (Kernklinikbereiche) erfasst (Falldefinition der ESCMID Study Group for Clostridium difficile) und als ambulant (innerhalb der ersten 3 Tage nach Aufnahme diagnostiziert und nicht in den letzten 4 Wochen stationär) oder nosokomial erworben klassifiziert. Die Analysen der Daten aus 2014 wurden nun für zwei Kliniken und jeweils mehrere Bereiche durchgeführt. Die monatlichen Inzidenzdichten (in Fälle pro 1000 Pflegetage) und Antibiotikaverbrauchsdichten (in DDD und RDD pro 100 Pflegetage) wurden ermittelt und bezüglich bestem Vorhersagemodell (Zeitreihen) analysiert. Als Kontrolle diente die ambulant erworbene CDAD. Ergebnisse Im ATHOS‐Bereich von Klinik A wurden 194 CDAD‐Fälle erfasst, darunter 148 nosokomiale Fälle (Inzidenzdichte 0.32/1000 im operativen und 0.65 im nicht‐operativen Bereich). In Klinik B waren es 245 Fälle, darunter 212 nosokomiale Fälle (Inzidenzdichte 0.82/1000 im operativen und 0.71/1000 im nicht‐operativen Bereich). Sowohl in den operativen als auch in den nicht‐operativen Bereichen ergab sich über den monatlichen Antibiotikaverbrauch (gemessen als Verbrauch von 3GC versus 1+2GC versus FQ versus Penicillinderivate) keine konsistente Prädiktion (Abbildungen 1 und 2). Diskussion Die nosokomiale CDAD‐Häufigkeit in Kliniken der Maximalversorgung scheint nicht sicher mit dem Einsatz von 3GC oder FQ zu korrelieren. Weitere Beobachtungsstudien und Interventionsstudien unter Einschluss von Kliniken mit größerer Varianz im Antibiotikaverbrauch sind sinnvoll, um das CDAD‐Präventionspotenzial mittels ABS‐Programmen besser beschreiben zu können. Figure 1 Figure 2 132 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 104 In vitro susceptibility to agents other than betalactams of third generation cephalosporin‐
resistant Enterobacteriaceae (3GCREB) isolated on hospital admission within the first ATHOS multicenter prevalence study A. Mischnik1, P. Baumert1, A. Hamprecht2, A. M. Rohde3, S. Feihl4, B. Obermann5, S. Peter6, A. Serr1 P. Gastmeier3, H. Seifert2, W. Kern1, U. D. ATHOS‐Studiengruppe7 1
Universitätsklinikum Freiburg, Freiburg, Germany Universitätsklinikum Köln, Cologne, Germany 3
Charitè, Berlin, Germany 4
TUM‐Klinikum Rechts der Isar, Munich, Germany 5
Universitätsklinikum Schleswig‐Holstein, Lübeck, Germany 6
Universitätsklinikum Tübingen, Tübingen, Germany 7
DZIF Deutsches Zentrum für Infektionsforschung, Cologne, Germany 2
Background The ATHOS study (antibiotic therapy optimisation study) is a multicentre study which evaluates the admission prevalence and incidence of multi‐drug resistant organisms and aims to implement antibiotic stewardship programs in the inpatient and outpatient setting. As part of this project, a first prevalence study was carried out in 2014 at six German university hospitals and included the assessment of admission prevalence of rectal third generation cephalosporin‐resistant Enterobacteriaceae (3GCREB). The aim of present study was to assess to evaluate the in vitro susceptibility of the admission prevalence 3GRCEB isolates to non‐betalactam antibiotics. Methods A total of 4376 adult patients from six university hospitals participated in 2014 survey. Rectal swabs or stool samples taken within 72 h of admission were positive for 3GCREB carriers in 9.5% with 417 isolates of which 343 Escherichia coli, 38 Klebsiella spp., 20 Enterobacter spp., and 11 other 3GCREB were available for MIC testing using microdilution assays (Merlin Diagnostika, Bornheim‐Hersel) according to standard procedures. More than 90% of the isolates were ESBL producers, most commonly of CTX‐M‐1 group (see abstract Hamprecht et al). Results Table 1 shows the rates of reduced susceptibility or resistance to a number of non‐betalactam agents at various breakpoints. There were major differences in non‐betalactam susceptibility patterns between ESBL‐positive E. coli and other 3GCREB isolated from adult patients on admission. Of note were the relatively low rate of reduced susceptibility and resistance to fluoroquinolones, gentamicin and chloramphenicol (in particular among Enterobacter spp.) and the relatively high rate of decreased susceptibility to rifaximin (in particular among species other than E. coli). High‐level fluoroquinolone resistance was relatively rare. As expected, high resistance rates were observed for macrolides and tetracycline (table 1), and very little resistance was observed for fosfomycin, amikacin and colistin (data not shown). Figure 1 Conclusion Among these 3GCREB admission isolates reflecting in part community colonization reduced fluoroquinolone susceptibility although <50% was more common in E. coli than in other 3GCREB, and high‐level fluoroquinolone resistance was still rare. Klebsiella and Enterobacter spp. showed high rates of reduced susceptibility to rifaximin and tigecycline. 133 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 105 A systematic analysis platform to elucidate antibiotic mechanisms A. Müller1, I. Engels1, M. Rausch1, F. Grein1, H.‐ G. Sahl2, T. Schneider1 1
2
Universität Bonn, Pharmazeutische Mikrobiologie, Bonn, Germany Universität Bonn, Medizinische Mikrobiologie, Bonn, Germany Novel antibiotics with unprecedented mechanisms of action are urgently needed to overcome rising resistance. Analysis of the mechanism of action of an antibiotic and identification of the molecular target are integral components of the drug development process. Without this detailed knowledge, rational drug design is strongly hampered. We built up a comprehensive mode of action (MoA) platform, combining whole cell screenings and a biochemical platform that allows rapid identification of antibiotic mechanisms and targets on all cellular levels. Initial screenings of compound and extract libraries in whole‐cell based assays identify biologically active compounds and provide first indications of the metabolic pathway affected. “Smart screens”, such as the antisense (AS) RNA technology, provide intrinsic information on the target structure. AS RNA mediated gene depletion confers selective antibiotic hypersensitivity by lowering the cellular pool of the cognate protein target. A biochemical analysis platform comprising more than 60 individual in vitro assays further allows for the identification and characterization of a specific target on the molecular level. 134 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 106 Analysis of Plasmid Diversity among Acinetobacter baumannii Outbreak Strains using Whole Genome Sequencing R. Auer1,2, M. Fritzenwanker1,2, Y. Yao1,2, L. Falgenhauer1,2, H. Ghosh1,2, H. Seifert3,4, C. Imirzalioglu1,2 T. Chakraborty1,2 1
Institut für Medizinische Mikrobiologie, Justus‐Liebig‐Universität Giessen, Giessen, Germany Deutsches Zentrum für Infektionsforschung (DZIF), Standort Giessen‐Marburg‐Langen, Giessen, Germany 3
Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Universität zu Köln, Cologne, Germany 4
Deutsches Zentrum für Infektionsforschung (DZIF), Standort Bonn‐Köln, Cologne, Germany 2
Introduction and purpose Acinetobacter baumannii is an increasingly important pathogen, mainly associated with hospital acquired pneumonia and the potential to cause nosocomial outbreaks. An integral part of this property might be its remarkable potential to acquire resistance mechanisms. The uptake of plasmids by horizontal gene transfer has been described as a mechanism for A. baumannii to acquire antibiotic resistance genes but their contribution to A. baumannii multidrug resistance has never been clearly evaluated. To address this aspect, we investigated the diversity of A. baumannii outbreak isolates and examined their contribution to antibiotic resistance. Methods 27 A. baumannii outbreak isolates, recovered between 2006 and 2015 in Germany, Poland and Sweden were sequenced using the Illumina MiSeq system. De novo assembly was performed using the CLC Genomics Workbench. Contigs of potential plasmids were manually joined using references from the NCBI database and mate pair information. Remaining plasmid contigs were concatenated to pseudogenomes. Annotation was performed using the PROKKA software and A. baumannii genomes were compared with GECO. Plasmids were assigned to different replicon types using the scheme of Bertini et al.1 Results The number of plasmids per isolate varied between 0 and 6, with a total number of 45 plasmids in 27 strains. Plasmids belonged to 16 different replicon types. Four plasmids could not be assigned to a replicon type. New replicon types were named RepN1‐8. The predominant replicon type was Aci1 (12 plasmids). None of the Aci1 plasmids contained antibiotic resistance genes. 10 isolates contained Aci6 plasmids, a conjugative plasmid carrying the aminoglycoside resistance gene aphA6 but only one of those encoded an additional blaOXA‐23‐like carbapenemase as previously described for Aci6 plasmids. In total, only 26 out of a total of 288 resistance genes were located on plasmids. Conclusion A. baumannii share a large plasmid population, with only a few common and a majority of rare plasmids. Although few resistance genes like the aphA6 are plasmid encoded, the majority of resistance genes are of chromosomal origin. 1
Bertini A et al.: Characterization and PCR‐based replicon typing of resistance plasmids in Acinetobacter baumannii. 135 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 107 Plasmid diversity and dynamics during a multi‐species nosocomial outbreak Y. Rezazadeh1,2, Y. Yao1,2, C. Imirzalioglu1,2, L. Falgenhauer1,2, J. Overmann3,4, B. Bunk3,4, A. Goesmann2,5 O. Schwengers2,5, T. Hain1,2, E. Domann1,2, T. Chakraborty1,2 1
Institute for Medical Microbiology, Justus‐Liebig‐University Giessen, Giessen, Germany German Center for Infection Research (DZIF), partner site Giessen‐Marburg‐Langen, Giessen, Germany 3
Leibniz Institute DSMZ‐Bioinformatics, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany 4
German Center for Infection Research (DZIF), partner site Hannover‐Braunschweig, Braunschweig, Germany 5
Bioinformatics Computational Biology, Justus‐Liebig‐University Giessen, Giessen, Germany 2
Prevalence of outbreaks of carbapenem‐resistant Enterobacteriaceae is an increasing cause of nosocomial infections. Between October 2013 and September 2014, an outbreak with 132 patients occurred in a single hospital in Southern Hesse, Germany. The outbreak was unusual in that it involved many different bacterial species and sub‐species of the genus Enterobactericeae all exhibiting carbapenem‐resistance. Hence plasmid‐ based transmission of resistance between bacteria of different species was suspected. S1‐PFGE revealed plasmid sizes of between 6.55 kb and 340 kb but indicated no commonly occurring plasmid in these isolates. We analysed 24 isolates from 19 patients and 12 environmental samples from the hospital kitchen and food by whole genome sequencing (Illumina MiSeq) and single‐molecule‐real‐time sequencing (SMRT, PacBio RSII). S1 nuclease digestion followed by pulsed‐field gel electrophoresis (S1‐PFGE) was carried out. Comparative analysis using ResFinder, GenDB and EDGAR and conjugation studies with E. coli J53 as recipient strain were conducted. All of 36 isolates belonging to 9 different species harboured the blaKPC‐2 gene in a 9,571 bp unique genetic environment on an IncN plasmid.1 However, the sizes of the IncN plasmids varied considerably in size, from 58,447 to 78,849 bp and harboured deletions and insertions probably caused by mobile genetic elements. The most frequently occurring plasmid comprises 78,021 bp encoding 100 CDS and carries 13 antibiotic resistance genes: three beta‐lactamases, four aminoglycoside resistance genes, as well as genes encoding resistance against quinolones, macrolides, rifampicin, chloramphenicol, sulphonamides and trimethoprim flanked by different insertion elements in a 40,501 bp region between the traI and traG genes. Plasmids were transferrable to E. coli J53 by conjugation but also showed size variation following transfer indicating that both mobility and genetic variability are inherent properties of these plasmids. Results from our study demonstrate that plasmid‐mediated multispecies based nosocomial outbreaks with carbapenem‐resistance are emerging threats that challenge current antibiotic therapy options. References [1] Yao Y. et al. Complete Nucleotide Sequence of a Citrobacter freundii Plasmid Carrying KPC‐2 in a Unique Genetic Environment; Genome Announcement 2014 Apr 24; 2(2). Pii: e00356‐14; doi:10.1128/genomeA.00356‐14 136 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 108 Molecular characterization of extended‐spectrum beta‐lactamase producing E. coli ST 131 from humans and companion animals in Germany H. Ghosh1, L. Falgenhauer1, J. Schmiedel1, K. Gentil1,2, Y. Yao1, C. Imirzalioglu1, T. Chakraborty1 1
Institute of Medical Microbiology, Justus‐Liebig‐University Giessen and German Center for Infection Research (DZIF), Partner site Giessen‐Marburg‐Langen, Germany, Giessen, Germany Multidrug‐resistant (MDR) Escherichia coli sequence type 131 (ST131) is disseminated worldwide and causes urinary tract and bloodstream infections in human and animals. E. coli ST131 are often associated with resistance to extended‐spectrum beta‐lactams (ESBL) thus offering only limited treatment options, as well as higher morbidity, mortality and health care costs. Therefore a better understanding of the molecular mechanisms that result in the epidemiological success of E. coli ST131 is required. This study investigated the prevalence and dissemination of MDR E.coli ST131 across different hosts in Germany. During the study period (2009‐2011), a total of 361 nonrepetitive ESBL‐producing E.coli were isolated from humans (n=183) and companion animals (n=178) from central Hesse in Germany. All isolates were subjected to antibiotic susceptibility testing. Preliminary presence of blaCTX‐M allele was confirmed by PCR. A subset of 102 isolates (n=52 human; n=50 animal) was analyzed using whole genome sequencing (WGS) (Illumina MiSeq platform). All isolates were screened for ST131 by in silico multi locus sequence typing. Core genome SNPs based phylogeny, virulome profile, antibiotic resistance gene profile, genomic islands, serotype, plasmidome, and phylogenetic groups were analyzed in silico. A total of eighteen (18%) isolates were identified to be ST131 E. coli by multilocus sequence typing, of which 77% (n=14) were of human and 27% (n=4) of animal origin. These isolates were serotyped as O25b:H4 (78%) and O16:H5 (22%). Based on their core genome profile, all the ST131 isolates belonged to the B2 phylo‐group which further divided into two sub‐lineages. Of the 18 isolates, 11 isolates harbored a CTX‐M‐15 beta‐
lactamase enzyme, 5 harbored CTX‐M‐1 and two strains harbored CTX‐M‐14 or CTX‐M‐27. CTX‐M‐15 was observed to be present on the chromosome as well as on plasmids, frequently in more than one locus. Human and animal isolates showed overlapping patterns of plasmid types, virulence genes and antimicrobial resistance genes profile. Core genome SNPs based phylogeny demonstrates a close relationship between human and animal isolates. In this study we observe highly clonal strains of E. coli ST131 persistent in humans as well as in animals. The clonal nature of whole genome is well supported at nucleotide levels. In addition, the plasmids present in the studied isolates were also clonal. Therefore, this study supports possible dissemination of E. coli ST131 between humans and animals. 137 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 109 Admission Prevalence of third generation cephalosporin‐resistant Enterobacteria (3GCREB): A cross‐sectional study in 6 university hospitals A. M. Rohde1, J. Zweigner1,2, M. Wiese‐Posselt1, A. Hamprecht2, F. Schwab1, W. Kern3, P. Gastmeier1, H. Seifert2 DZIF‐ATHOS Study Group1,2,3,4,5,6 1
Charité Berlin, Institut für Hygiene und Umweltmedizin, Berlin, Germany University Hospital Cologne, Institute of Medical Microbiology, Immunology and Hygiene, Cologne, Germany 3
University Hospital Freiburg, Departement for Infectiology, Freiburg, Germany 4
University Hospital Tübingen, Tübingen, Germany 5
University Hospital Klinikum Rechts der Isar, Munich, Germany 6
University Hospital Schleswig‐Hostein, Lübeck, Germany 2
Introduction This admission prevalence survey is part of the multicenter study ATHOS (antibiotic therapy optimisation study). ATHOS aims at collecting prevalence and incidence data for nosocomial carriage of multi‐drug resistant organisms (MDROs) and to intervene in the inpatient and outpatient setting. Objectives The aim of this admission prevalence survey was to assess the rectal carriage of third generation cephalosporin‐resistant enterobacteria (3GCREB) in patients on hospital admission and to perform risk factor analyses for 3GCREB carriage. Methods In 2014, we recruited adult patients within 72 h of admission to non‐intensive care units in six German university hospitals. We obtained rectal swabs that were screened for 3GCREB. Each patient was asked to answer a short questionnaire on potential risk factors for colonisation with MDROs. Univariable and multivariable risk factor analyses were performed on preliminary data to identify those factors that were associated with 3GCREB prevalence. Results Of the 4376 patients included, 417 patients were 3GCREB carriers (admission prevalence of 9.5%). Surprisingly, 42.2% of all 3GCREB isolates were additionally resistant to fluoroquinolones. Five patients (1.2%) were colonised with carbapenemase‐producing enterobacteria. Multivariable analysis associated the following risk factors with 3GCREB colonisation: centre, previous MDRO colonisation (OR = 2.1, p<0.001), antibiotic use (OR=2.1, p<0.001), travel outside of Europe (OR=2.9, p<0.001), occupational animal contact (OR=1.3, p=0.033) and management of gastroesophageal reflux disease (GERD) (OR=1.2, p=0.010). Conclusion To our knowledge, this is one of the largest admission prevalence surveys of 3GCREB in Germany. Interestingly, occupational animal contact, medical management of GERD and the specific centres to which the patients where admitted proved to be additional risk factors for 3GCREB colonisation on hospital admission. Whether information present on admission will be useful to improve prediction of nosocomial colonisation and infection as well as target infection control measures and therapy needs to be determined. 138 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 110 Sensitizing Staphylococcus aureus to cationic antimicrobial peptides by MprF‐inhibiting monoclonal antibodies C. Slavetinsky1, A. Peschel1 1
Interfaculty Institute of Microbiology and Infection Medicine, Tübingen University, German Center for Infection Research, partner site Tübingen, Tübingen, Germany The lysinylation of negatively charged phosphatidylglycerol by MprF (the Multiple peptide resistance Factor) confers resistance to cationic antimicrobial peptides (CAMPs), several antibiotics (e.g. daptomycin) and the host immune system in general (1). MprF of Staphylococcus aureus is a bifunctional enzyme consisting of separable domains for lysyl‐phosphatidylglycerol (Lys‐PG) production and Lys‐PG flipping (2). As MprF proteins are crucial virulence factors for various bacterial pathogens and play a major role in the emerging resistance against the blockbuster antibiotic daptomycin, they represent an attractive target for novel anti‐virulence drugs. We designed several monoclonal antibodies in order to block MprF and analyzed their capacity to support the killing of S. aureus by CAMPs, antibiotics, and by human neutrophils. We found that those antibodies block the flippase reaction of MprF and thereby render S. aureus more susceptible to clearance by the host immune system and antibiotics. Most notably they restore the susceptibility of a daptomycin‐resistant clinical isolate and prevent growth of USA300 under subinhibitory concentrations of antibiotics. These findings provide a promising new approach for anti‐virulence therapy against bacteria based on pathogen sensitization to the host immune system or antibiotics. Furthermore, they offer interesting options for studying the MprF mode of action and its role in daptomycin resistance. Keywords MprF, CAMPs, daptomycin resistance, monoclonal antibodies, anti‐virulence drugs 1. Peschel A, Jack RW, Otto M, Collins LV, Staubitz P, Nicholson G, Kalbacher H, Nieuwenhuizen WF, Jung G, Tarkowski A, van Kessel KP, van Strijp JA. The Journal of experimental medicine. 2001. 193:1067‐1076. 2. Ernst CM, Staubitz P, Mishra NN, Yang SJ, Hornig G, Kalbacher H, Bayer AS, Kraus D, Peschel A. PLoS Pathog. 2009. 5:e1000660. 139 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 111 Detection of methicillin resistant coagulase‐negative staphylococci by MALDI‐TOF MS D. Schuster1, M. Josten1, I. Bodenstein1, E. Sib1, M. Gajdiss1, H.‐ G. Sahl1, G. Bierbaum1 1
Universität Bonn, Bonn, Germany Introduction Methicillin resistant coagulase‐negative staphylococci are frequently isolated in hospitals, where they cause nosocomial infections. A high percentage of these strains is resistant to methicillin. S. epidermidis is encountered most frequently, but others as e. g. S. lugdunensis have been reported to be even more pathogenic (1). A peptide (PSM‐mec) is encoded on the type II, III and VIII SCCmec cassettes in the genomes of nosocomial methicillin‐resistant Staphylococcus aureus (MRSA) strains. Agr‐positive MRSA that possess these SCC‐mec cassettes excrete this peptide and we have shown in previous work that it is visible during matrix‐
assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF MS) of whole cells (2). By analysing the spectra, it is possible to identify MRSA already during mass spectrometry by the presence of a signal at m/z 2415. Since similar SCCmec cassettes have been acquired by coagulase‐negative species, we have evaluated a collection of clinical coagulase‐negative staphylococci for the presence of the structural gene encoding PSM‐mec by PCR and the appearance of the corresponding signal during mass spectroscopy. Materials and Methods So far, more than 350 strains have been characterized, among them 73 % S. epidermidis, but also including S. haemolyticus (10 %), S. hominis (9 %), S. capitis, S. lugdunensis, S. simulans, S. warneri, S. schleiferi, S. caprae, S. auricularis and S. cohnii. The cells were smeared onto the target and analysed directly or after extraction on the target as described in (2). Results 76 % of all isolates were methicillin‐resistant, however, only 27 % of all isolates (or 35 % of all resistant isolates) contained the gene psm‐mec. In MALDI‐TOF MS spectra, 83 % of the strains that harbored the gene yielded the correct signal. There were no false positive results. Conclusion The results show that the peak at m/z 2415 signals methicillin resistance in those coagulase‐negative staphylococcal species that were analysed here. (1) Argemi et al., J. Clin. Microbiol. 2015 (7):2030‐6. (2) Josten et al., Int. J. Med. Microbiol. 2014 (8):1018‐23. 140 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 112 An abundance of antimicrobial substances governs microbial competition in the human nasal microbiota B. Krismer1, D. Janek1, A. Peschel1, A. Kulik1 1
Universität Tübingen, Institut für Infektionsbiologie, Tübingen, Germany The human nasal microbiota is highly variable and dynamic often enclosing major pathogens such as Staphylococcus aureus. The potential roles of bacteriocins or other mechanisms allowing certain bacterial clones to prevail in this nutrient‐poor habitat have hardly been studied. Of 90 nasal staphylococcal strains, unexpectedly, the vast majority (82%) was found to produce antimicrobial substances in particular under habitat‐specific stress conditions. Activity spectra were generally narrow but highly variable with activities against certain Gram‐positive, Gram‐negative, or both groups of bacteria. A representative bacteriocin was identified as a nukacin‐related peptide whose inactivation strongly reduced the producer's capacity to limit growth of other nasal bacteria. Of note, the bacteriocin genes were found on mobile genetic elements exhibiting signs of extensive horizontal gene transfer and recombination events. Thus, continuously evolving bacteriocins appear to govern bacterial competition in the human nose and specific bacteriocins may become important agents for eradication of notorious endogenous pathogens. 141 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 113 Sensitizing Staphylococcus aureus to cationic anti‐microbial peptides A. Jorge1,2, U. Bilitewski3,4, A. Peschel1,2 1
University of Tübingen, Interfaculty Institute of Microbiology and Infection Medicine, Tübingen, Germany German Center for Infection Research, Tübingen, Germany 3
Helmholtz Centre for Infection Research, Braunschweig, Germany 4
German Center for Infection Research, Hannover‐Braunschweig, Germany 2
Bacteria that colonize and infect the human body have adapted well to our immune defenses. The particular case of Staphylococcus aureus is noteworthy as it can be a normal commensal but also a pathogen. Indeed, S. aureus developed mechanisms to resist to the host immune system, in particular, to cationic antimicrobial peptides (CAMPs). With a general negatively charged surface, S. aureus repulses CAMPs by decorating the cell surface with positively (or neutral) residues. As examples are: a) the incorporation of L‐lys to phosphatidylglycerol by MprF; b) the D‐alanylation of teichoic acids by DltA‐D; c) the amidation of the peptidoglycan by GatD/MurT. Accordingly, S. aureus mutants of these proteins are more susceptible to CAMPs and have reduced capacity to cause infections in animal models. We focus on the screening of molecules that sensitize S. aureus or other healthcare‐associated pathogens to CAMPs. Targeting non‐essential proteins is thought to result in lower emergence of resistance and thus having a better outcome for clinical use. Our approach includes the growth of bacteria with non‐inhibitory concentration of a CAMP‐like antibiotic (daptomycin) together with a library molecule. Compounds that inhibit targets involved in CAMPs resistance will affect bacterial susceptibility to daptomycin and growth will be reduced. Three unrelated libraries were screened: a) 143 natural compounds from myxobacteria provided by the Helmholtz Institute for Pharmaceutical Research Saarland; b) the VARious sources of synthetic compounds Library (VAR) comprising 3344 compounds and c) the Library of Pharmacologically Active compounds (LOPAC) with 1408 drugs. From the myxobacteria library, myxovirescin A was identified as an interesting candidate. We show that S. aureus is more susceptible to non‐inhibitory concentrations of several cationic antimicrobials, such as daptomycin, polymyxin B, and gallidermin only in the presence of myxovirescin A and not of other type of antibiotics. Myxovirescin has been found to inhibit the bacterial lipoprotein signal peptidase II (Lsp). We confirmed that inhibition in S. aureus but Lsp inhibition did not led to susceptibility to CAMPs suggesting that myxovirescin A may have one of the CAMP resistance proteins as a second target. High‐throughput screening of the VAR library resulted in 28 putative hits. From these, only 14 showed concentration‐dependent activity and will be further studied. From the LOPAC library around 35 drugs were synergistic with daptomycin and the vast majority were dopamine‐receptor antagonists. The relationship between these antagonists and daptomycin susceptibility is under investigation. Taken together our strategy allowed us to identify around 50 putative compounds (1% of the total amount of molecules screened) that are under further analysis to specifically look for inhibitors of proteins involved in CAMPs resistance. 142 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 114 DZIF TI Bioinformatics Platform ‐ Giessen‐Marburg‐Langen Site ‐ O. Schwengers1,2, A. Schaprian1,2, R. Hilker1,2, Y. Yao1, T. Hain1, A. Goesmann2, T. Chakraborty1 1
2
Justus‐Liebig‐Universität Giessen, Institut für Medizinische Mikrobiologie, Giessen, Germany Justus‐Liebig‐Universität Giessen, Bioinformatik und Systembiologie, Giessen, Germany The Giessen‐Marburg‐Langen partner site of the TI Bioinformatics platform provides various bioinformatic services mainly focusing on the large‐scale analysis of prokaryotic data sets generated by next generation sequencing and ‐omics technologies. Activities range from the development of new user‐friendly software solutions to tailored bioinformatics support in dedicated cooperation projects, creation of project specific databases, hands‐on support and advice on data analysis. The in‐house developed tools GenDB, EDGAR and ReadXplorer address these issues and facilitate automatic annotations of prokaryotic genomes, comparative analyses as well as visualizations of mapped reads each providing a versatile graphical user interface. Training workshops are offered for clinicians and biologists, improving literacy and skills in computational data analysis required for their specific scientific projects. Another major goal is the development of sophisticated pipelines and their scale‐up to meet dynamically growing requirements and data throughput. To this end, the Giessen‐Marburg‐Langen partner site operates and provides a high‐performance compute cluster. 143 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 115 Discrimination of Escherichia coli isolates by MALDI‐TOF mass spectrometry P. Thomas‐Morr1, H. Rohde1, L. M. Biehl2, G. Bierbaum3, M. Christner1 1
Universitätsklinikum Hamburg‐Eppendorf, Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Hamburg, Germany 2
Universitätsklinikum Köln, Klinik für Innere Medizin, Cologne, Germany 3
Universitätsklinikum Bonn, Institut für Medizinische Mikrobiologie, Immunologie und Parasitologie, Bonn, Germany MALDI‐TOF mass spectrometry fingerprinting is now widely used for routine identification of cultured microorganisms. Commercially available systems provide reliable species level resolution within most clinically relevant genera and several proof‐of‐concept studies have achieved various degrees of subspecies level discrimination. However, the techniques’ applicability for epidemiological purpose has been questioned due to lack of reproducibility and discriminatory power. In order to investigate the usefulness of MALDI‐TOF mass spectrometry for subspecies level discrimination of Escherichia coli we’ve investigated the spectral diversity within a collection of > 200 clinical E. coli isolates and systematically addressed genotype‐phenotype correlations in a collection of > 3500 knockout strains and > 80 plasmid transductants. Our assessment of endemic E. coli isolates revealed a high degree of spectral diversity within endemic E. coli isolates. The majority of isolates could be reliably discriminated from > 90% of the remaining population by various algorithms in inter‐assay comparisons. However, further increase in discriminatory power required strict standardization of testing conditions which might be difficult to achieve between operators or laboratories. Our analysis of plasmid transductants revealed several transducible markers which were in part shared between different plasmids. Finally, several E. coli peaks could be annotated from knockout mutant spectra, facilitating the assessment of population spectral diversity using available genome sequence data. The resolution of MALDI‐TOF mass spectrometry might be insufficient to challenge established typing systems. However, cost and throughput still limit the applicability of sequence based typing in a number of cases. The substantial spectral variability observed within E. coli isolates could e. g. allow for outbreak strain screening in large strain collections or as part of the species level identification workflow in clinical laboratories. 144 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 116 Preclinical development of HY‐133, a recombinant phage lysin for rapid decolonization of Staphylococcus aureus from nasal habitats E. Idelevich1, A. Scherzinger2, B. Krismer3,4, C. Weidenmaier3,4, L. Heitmann5, G. Winter6,7, T. Hesterkamp5 W. Mutter2, K. Becker1, A. Peschel3,4 1
University Hospital Münster, Institute of Medical Microbiology, Münster, Germany Hyglos GmbH, Bernried, Germany 3
Universit of Tübingen, Interfaculty Institute of Microbiology and Infection Medicine, Tübingen, Germany 4
German Center for Infection Research, Partner Site Tübingen, Tübingen, Germany 5
German Center for Infection Research, Pertner site Hannover‐Braunschweig, Braunschweig, Germany 6
Ludwig‐Maximilian University Munich, Pharmaceutical Technology and Biopharmacy, Munich, Germany 7
German Center for Infection Research, Partner Site Munich, Munich, Germany 2
Current strategies to eradicate antibiotic‐resistant pathogens from the microbiomes of risk patients are scant. Available approaches rely on broad‐spectrum antibiotics e.g. for eradication of Staphylococcus aureus from human noses with the antibiotic mupirocin. Because general inhibition of the microbiota bears severe risks of resistance development, outgrowth of other pathogens, or rapid recolonization by the same pathogen, highly selective, yet efficient and rapid eradication strategies are urgently needed. Along this line, bactericidal proteins derived from bacteriophages are discussed as alternative antiinfective strategies. They combine very fast bactericidal properties, stability and activity at various environmental conditions, and high selectivity to a particular bacterial target species. Specific lytic phage proteins could enable new, highly effective eradication strategies to overcome increasing mupirocin resistance (MupR) and simplify the complicated and lengthy mupirocin treatment regimen. During the first funding period, we developed engineered lytic proteins derived from the S. aureus phage K and the bacteriocin lysostaphin, which were found to act very efficiently against S. aureus including diverse methicillin‐resistant S. aureus (MRSA) lineages while clinical strains of coagulase‐
negative staphylococci were not affected. Our optimized product candidate HY‐133 was engineered to specifically address unmet medical needs for a decolonization agent that is (i) highly efficient, acting fast in a bactericidal fashion, ideally within 24 hours prior to or subsequent to hospital admission, (ii) resistance‐
breaking to MupR isolates and itself subject to low rates of resistance formation, and (iii) highly species‐specific for S. aureus, both methicillin‐susceptible S. aureus (MSSA) and MRSA, without influence on the co‐colonizing nasal microbiome as a bias for rapid re‐ colonization by S. aureus. HY‐133 was effective in an animal model and funding for its GMP‐grade production, formulation in an application‐friendly and stable dosage form, and toxicological evaluation from DZIF Flexible Funds has recently been approved. We envisage evaluating HY‐133 in a clinical phase I/IIa study and developing it into a therapeutic alternative to mupirocin. Selective pathogen decolonization by specific lytic phage proteins could become important strategies against several other antibiotic‐resistant endogenous pathogens. 145 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 117 Biosynthetic Studies of Telomycin Reveal New Lipopeptides with Enhanced Activity C. Fu1, L. Keller1,2, A. Bauer3, M. Brönstrup2,4, A. Froidbise5, P. Hammann6, J. Herrmann1,2, G. Mondesert5 M. Kurz3, M. Schiell3, D. Schummer3, L. Toti3, J. Wink2,4, R. Müller1,2 1
Helmholtz Institute for Pharmaceutical Research Saarland, Microbial Natural Products, Saarbrücken, Germany German Centre for Infection Research, Braunschweig, Germany 3
Sanofi‐Aventis Deutschland GmbH, R&D LGCR, Frankfurt am Main, Germany 4
Helmholtz Centre for Infection Research, Braunschweig, Germany 5
Sanofi R&D, Toulouse, France 6
Sanofi‐Aventis Deutschland GmbH, Frankfurt am Main, Germany 2
Telomycin (TEM) is a cyclic depsipeptide antibiotic active against Gram‐positive bacteria. In this study, five new natural telomycin analogues produced by Streptomyces canus ATCC 12646 were identified. To understand the biosynthetic machinery of telomycin and to generate more analogues by pathway engineering, the TEM biosynthesis gene cluster has been characterized from S. canus ATCC 12646: it spans approximately 80.5 kb and consists of 34 genes encoding fatty acid ligase, nonribosomal peptide synthetases (NRPSs), regulators, transporters and tailoring enzymes. The gene cluster was heterologously expressed in Streptomyces albus J1074 setting the stage for convenient biosynthetic engineering, mutasynthesis and production optimization. Moreover, in‐frame deletions of one hydroxylase and two P450 monooxygenase genes resulted in the production of novel telomycin derivatives, revealing these genes to be responsible for the specific modification by hydroxylation of three amino acids found in the TEM backbone. Surprisingly, natural lipopeptide telomycin precursors were identified when characterizing an unusual precursor deacylation mechanism during telomycin maturation. By in vivo gene inactivation and in vitro biochemical characterization of the recombinant enzyme Tem25, the maturation process was shown to involve the cleavage of previously unknown telomycin precursor‐
lipopeptides, to yield 6‐methylheptanoic acid and telomycins. These lipopeptides were isolated from an inactivation mutant of tem25 encoding a (de)acylase, structurally elucidated and then shown to be deacylated by recombinant Tem25. The TEM precursor and several semisynthetic lipopeptide TEM derivatives showed rapid bactericidal killing and were active against several multidrug‐resistant (MDR) Gram‐positive pathogens, opening the path to future chemical optimization of telomycin for pharmaceutical application. Figure 1 146 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 118 Identification of novel inhibitors targeting colonization and virulence of gastrointestinal pathogens: Inhibition of the biogenesis of outer‐membrane virulence factors J. M. Schweers1, M. Buhl1, U. Bilitewski2, I. Autenrieth1, M. Schütz1 1
2
Eberhard Karls University, Medical Microbiology, Tübingen, Germany Helmholtz Centre for Infectio Research, Braunschweig, Germany Despite the fact, that the enormous economic burden and individual suffering caused by gastrointestinal infections permanently persists in developing and newly industrialized countries, healthcare systems in First world countries underestimated its significance for a long time. The alarming prevalence of multidrug‐resistant gram‐negative bacteria, combined with a high epidemic potential of gastrointestinal pathogens, however, demonstrates the urgent need for new antibiotics and antiinfectives worldwide. 2,5 million deaths per year were actually caused by acute diarrheal infections. The most common causative agents of acute diarrheal infections, amongst others, are Yersinia enterocolitica, Campylobacter jejuni, Salmonella spp., Shigella spp., Escherichia coli, Vibrio cholerae, and Clostridium difficile. The established treatment based on antibiotics is mostly ineffective or may even have adverse side effects and result in prolonged shedding. In either way, antibiotic treatment also eradicates at least parts of the intestinal microbiome, and thereby disrupts colonization resistance, fosters overgrowth of pathogens and prolongs shedding times. Therefore, the development of future drugs should be focused on highly specific antiinfectives, which enable a direct pathogen‐specific treatment. One very promising strategy is the inhibition of the biogenesis of outer membrane virulence factors. Due to the fact that many decisive virulence‐associated outer membrane proteins (OMPs) of gram‐negative enteropathogens are substrates of the periplasmic chaperone SurA exclusively, we developed a new assay format to determine SurA in vitro chaperone activity. Previous publications by Behrens et al., 2001 and Buchner et al., 1998 documented an assay to determine SurA in vitro chaperone activity with extremely limited sensitivity and minimal detectable concentration, which was not suitable for high throughput screening (HTS). We now developed a luciferase‐based screening assay. This highly sensitive and robust test system has been validated extensively and now gives reliable output with an appreciable z‐factor of > 0,6. In cooperation with the HZI Braunschweig (Germany and the HZI Saarbrücken (Germany, we were able to screen over 8000 purified compounds and over 1000 extracts of myxobacteria. During the ongoing screening period, the assay generated four validated primary actives, which corresponds to a positive hit rate of 0,05 %. Additionally, we developed an elaborate follow‐up strategy to validate positive hits, which includes a well‐
established mouse infection model. We are looking forward to escalate our screening efforts and would like to use this abstract to invite all scientist who are interested in testing compound/natural extract libraries for an activity against the target structure SurA. 147 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 119 Genome Mining for Novel Antiinfectives M. Alanjari1, M. Adamek1, K. Steinke1, N. Ziemert1 1
DZIF, University Tübingen, Applied Natural Product Genome Mining, Tübingen, Germany Next generation sequencing methods have made sequencing faster, cheaper, and easier than ever and have revolutionized almost every field of biology. The constantly growing volume of DNA sequence data has made genome mining an important tool for the detection and prediction of promising secondary metabolites and has lead to a renaissance in natural product based drug discovery. Accurate bioinformatic analyses triage promising pathways, guide laborious wet lab experiments, and assist with the dereplication of already known compounds. Here we demonstrate how bioinformatic tools and comparative genomics can be used for a rapid and automated identification and examination of novel biosynthetic gene clusters and highlight first results of mining rare and underexplored actinomycete genera for promising natural compounds. 148 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 120 Antibiotic resistance‐modifying agents from marine‐derived fungi P. Barac1, H. Harms1, I. Engels2, T. Schneider2, G. M. König1 1
2
University of Bonn, Institute of Pharmaceutical Biology, Bonn, Germany University of Bonn, Institute for Pharmaceutical Microbiology, Bonn, Germany Antibiotic resistance development is an urgent world health concern aggravated by a lack of novel antibiotic discovery. ß‐lactams still represent the cornerstone for treating bacterial infections, but they are rapidly rendered useless due to antibiotic resistance. Since most of the antibiotics are natural products, natural sources might offer relevant solutions for this crisis. We target on marine fungi for finding new anti‐infectious compounds. The latter are often associated with algae, sponges, etc., and capable of producing interesting bioactive compounds to fight competitors. This project is focused on a special class of such compounds, termed resistance‐modifying agents (RMAs). When applied together with antibiotics, RMAs show a synergic effect, e.g. by interfering with the targets' resistance mechanism (ex: ß‐lactamases). Xanthocillin monomethyl ether (XMME) was found to be a RMA. It was isolated by activity‐guided fractionation from the fungus Dichotomomyces cejpii, associated with a tropical sponge. XMME showed intrinsic in vitro activity towards Staphylococcus aureus MSSA (MIC value= 2.5 µg/ml) and MRSA (MIC value= 20 µg/ml) strains. When used in combination with ertapenem (4 mg/ml), the latter being inactive towards the MRSA strain, extraordinary synergy was observed, resulting in a MIC value of <0.001 µg/ml. This result showed that XMME is a remarkable RMA. Further studies are needed to elucidate the exact mechanism of action for XMME. This study illustrates that developing assays and techniques to isolate and test the RMA activity of marine fungal metabolites does offer new perspectives in regaining efficiency for some of our most important antibiotics. Acknowledgements This research is funded by DZIF, Deutsches Zentrum für Infektionsforschung, Germany and supported by the BIGS DrugS, Bonn International Graduate School of Drug Sciences, Germany. 149 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 121 09.802: New antibiotics using novel methods of genome mining and heterologous expression of engineered gene clusters from Streptomyces collinus Tü 365 D. Eftime1, E. Stegmann1, T. Weber2,3, W. Wohlleben1 1
Universität Tübingen, IMIT Mikrobiologie/Biotechnologie, Tübingen, Germany Universität, Biologie, Hørsholm, Denmark 3
Technical University of Denmark, Novo Nordisk Foundation Center for Biosustainability, Hørsholm, Denmark 2
The aim of our project is the isolation and characterisation of new compounds, which can be used as lead structures for the development of novel antiinfections. The application of the bioinformatic tool antiSMASH resulted in the identification of different interesting silent biosynthetic gene clusters. Using new superhost strains and constitutive and inducible promoters, we succeeded in efficient expression and yield optimization. Exemplary, we present here the results obtained for Streptomyces collinus Tü 365, a strain, isolated 1972 from Kouroussa (Guinea). It is best known as producer of the antibiotic kirromycin, an inhibitor of the protein biosynthesis interacting with elongation factor EF‐Tu. Genome Mining revealed the presence 32 gene clusters encoding the biosynthesis of secondary metabolites in the genome of S. collinus Tü 365, indicating an enormous biosynthetic potential of this strain. The structural diversity of secondary metabolisms predicted for S. collinus Tü 365 includes PKS, NRPS, PKS‐NRPS hybrids, a lanthipeptide, terpenes and siderophores. While some of these gene clusters were found to contain genes related to known secondary metabolites, which also could be detected in HPLC‐MS analyses, most of the uncharacterized gene clusters are not expressed under standard laboratory conditions. We were able to make use of gene clusters, which previously have not been described for this strain and to connect the gene clusters to their respective biosynthesis products, thus a number of novel compound including a lanthipeptide, a carotenoide, four terpenoid compounds, an ectoine, a siderophore were characterized. 150 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 122 Development of Corallopyronin A to GLP pre‐clinical testing A. Schiefer1, K. Pfarr1, S. Bouhired1, G. M. König2, A. Hörauf1 1
2
Universitätsklinikum Bonn, Institut für med.Mikrobiologie, Immunologie und Parasitologie, Bonn, Germany Universität Bonn, Institut für Pharmazeutische Biologie, Bonn, Germany More than 150 million people in developing countries are infected with filarial nematodes that can cause lymphatic filariasis (Elephantiasis) or onchocerciasis (River Blindness). Filarial infections can be treated with antibiotics (e.g. doxycycline or rifampicin) that deplete the essential Wolbachia endobacteria, blocking worm development and causing worm death. Doxycycline cannot be used for children nor pregnant/nursing women, the use of rifampicin may increase the chances of resistance in TB, thus alternative antibiotics are needed. Corallopyronin A (CorA), produced by Corallococcus coralloides, inhibits the bacterial DNA dependent RNA polymerase but has an MoA different from rifampicin, thus it is active against Gram‐positive bacteria, including rifampicin‐resistant S. aureus, but is not effective against mycobacteria, thus would not drive resistance in TB‐
co‐infected filariasis patients. Within DZIF we have shown that CorA crosses many barriers to reach its intracellular target and depletes Wolbachia from infected insect cells and from Litomosoides sigmodontis infected BALB/c mice. In this model we showed that CorA inhibits development from the L4 stage into the adults. To determine macrofilaricidal activity, adult L. sigmodontis worms were treated ex vivo with CorA or doxycycline. The motility of the CorA treated worms decreased in a concentration dependent manner, faster than in the doxycycline treated worms. The macrofilaricidal effect of CorA was also shown using infected Mongolian Gerbils (Jirds) as a therapeutic treatment model. The Jirds were treated with antibiotics once microfilariae (MF) were detected in the blood (~12 weeks post infection). Eight weeks post treatment with CorA or doxycycline, no Wolbachia were detected in the MF. After 17 weeks, no MF could be found in the blood from the CorA treated Jirds, indicating worm sterility. The doxycycline treated Jirds achieved this effect only later, at 24 weeks post treatment. Both treatments strongly reduced the number of adult worms at 32 weeks post treatment. These results demonstrate that CorA is macrofilaricidal. Thus, CorA has potential as a new treatment option for human filarial infections without driving resistance to other RNAP inhibitors. To prepare CorA for use in humans, our project will complete toxicity studies in rodents and develop a CorA Galenic. This will provide essential guidance in planning and conducting GLP pre‐clinical studies with a pharmaceutical partner. 151 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 123 Evaluation of drug efficacy against intracellular‐ replicating Francisella tularensis H. von Buttlar1, M. Weis1, M. Antwerpen2 1
2
Institut für Mikrobiologie der Bundeswehr, Bakteriologie & Toxinologie, Munich, Germany Institut für Mikrobiologie der Bundeswehr, FMG, Munich, Germany Francisella tularensis is the causative agent of the zoonotic disease tularemia. The facultative intracellular gram‐negative bacterium naturally occurs in the northern hemisphere and is classified by the CDC as a category A agent, due to its high pathogenicity and low infection dose .Oone of its highly virulent subspecies, Francisella tularensis tularensis, can cause mortalities up to 60% in untreated patients and resistances against common antibiotics have been already observed. Therefore, the development and testing of new anti‐Francisella treatments is of major importance. After infection Francisella tularensis is able to proliferate in the cytoplasm of infected host cells, e.g. epithelial cells and especially macrophages. In order to measure the effectiveness of medical countermeasures during this intracellular profliferation, a standarizable system is needed. Macrophages (J774 and PMA‐differentiated THP‐1) were infected with Francisella tularensis and extracellular bacteria were killed by incubation with gentamycin subsequently. After 24h the efficacy of drug treatment against intracellularic Francisella was assessed by determing the bacterial load of the cells using two different immunofluorescence techniques. Besides fluorescence microscopy flow cytometry was used to gain a semi‐automated analysis system. Since Francisella tularensis gamma‐glutamyl‐ transpeptidase (gGT) is an essential enzyme for intracellular growth, we tested additionally gGT inhibitors for their anti‐Francisella function and could show a dose dependent reduction of intracellular Francisella proliferation. Thus, we established a useful standardized system for the verification of the efficacy of candidate drugs against Francisella tularensis during the intracellular phase of its life cycle. 152 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 124 Repurposing of Approved Drugs Targeting Cellular Proteins Involved in the Influenza Life Cycle T. Enkirch1, S. Sauber1, E. Gan2, S. Maurer‐Stroh3, V. von Messling2,1 1
Paul‐Ehrlich‐Institut, Veterinary Medicine, Langen, Germany Duke‐NUS Graduate Medical School, Emerging Infectious Diseases Program, Singapore, Singapore 3
Agency for Science, Technology and Research, Bioinformatics Institute, Singapore, Singapore 2
Background Influenza A virus is one of the most important causes of respiratory infections worldwide. In addition to annual epidemics, the emergence of new antigenic variants can result in pandemics with increased morbidity and mortality. Since resistance mutations rapidly emerge for drugs targeting viral proteins, this project aimed at assessing the antiviral activity of already approved drugs known to target cellular proteins that are involved in the influenza virus life cycle. Methods The drugs were initially screened for their ability to inhibit the replication of the mouse‐adapted influenza strain A/Puerto Rico/8/34 (PR8) in Madin‐Darby canine kidney cells. The proportion of infected cells after treatment was then quantified by flow cytometry to determine the therapeutic window. Candidates that reduced infection at least tenfold were further evaluated in vivo. Towards this, mice were treated orally starting one day before infection with PR8 and continuing for four days. Three days post‐infection, the animals were sacrificed and the viral load in the lung was compared to untreated controls. Furthermore, the efficacy of the most promising candidate was evaluated against a seasonal influenza strain in ferrets. Results Out of 15 compounds, four were able to inhibit infection ten to hundred fold without causing toxicity in vitro. Three of the drugs, naltrexon, ketotifen, and dextromethorphan resulted in 80 % reduction of infection, not only for PR8, but also for seasonal H1N1 and H3N2 strains. Dextromethorphan and to a lesser extent ketotifen also resulted in a significant reduction of lung titers, similar to the results obtained with amantadine, an approved influenza inhibitor. Subsequent evaluation of dextromethorphan in ferrets demonstrated no effect on viral load or fever severity but a reduction in clinical disease severity. Conclusions This proof‐of‐concept study constitutes a first step towards the use of drugs targeting cellular proteins to mitigate influenza and possibly other viral diseases. 153 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 125 Elucidation of the mechanism of action of teixobactin, a novel antibiotic isolated from uncultured bacteria I. Engels1, A. Müller1, T. F. Schäberle2, L. L. Ling3, L. Kim 4, T. Schneider1 1
Universität Bonn, Pharmazeutische Mikrobiologie, Bonn, Germany Universität Bonn, Pharmazeutische Biologie, Bonn, Germany 3
NovoBiotic Pharmaceuticals, Cambridge, USA 4
Antimicrobial Discovery Center, Northeastern University, Department of Biology, Boston, USA 2
Antibiotic resistance is one of the most serious health threats and the therapeutic options to treat infections caused by multi‐drug resistant strains are increasingly compromised. To combat antibiotic resistance novel antibiotics with unprecedented mechanisms of action are urgently needed. Since the discovery of penicillin, bacterial cell wall biosynthesis still remains a most attractive antibiotic target. Here we report the discovery of a novel cell wall biosynthesis inhibitor isolated from previously uncultured bacteria. Teixobactin, produced by Eleftheria terrae, is a 1.242 kDa depsipeptide containing unusual amino acids, like enduracididine and methylphenylalanine. In vivo and in vitro approaches revealed that the compound kills gram‐positive pathogens by binding to the highly conserved cell wall building block lipid II. In depth biochemical analysis further showed that teixobactin forms stable complexes with additional non‐protein targets, such as undecaprenyl‐PP‐sugar linkage units of wall teichoic acid and capsule precursors. (1) Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A, Schäberle TF, Hughes DE, Epstein S, Jones M, Lazarides L, Steadman VA, Cohen DR, Felix CR, Fetterman KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K. A new antibiotic kills pathogens without detectable resistance. Nature. 2015 Jan 7. doi: 10.1038/nature14098. 154 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 126 Genome mining‐guided drug discovery: new avenues to protease and proteasome inhibitors from bacterial sources L. Kaysser1, J. Zettler1, F. Leipoldt1, A. Baulig1, F. Wolf1 1
Universität Tübingen, Tübingen, Germany Proteases have been established as key factors in the pathogenesis of many parasitic protozoa and infectious bacteria. They are interesting targets for the development of new anti‐infective agents for a variety of different diseases e.g. malaria, sleeping sickness, Chagas disease and leishmaniasis but also tuberculosis. Terrestrial filamentous microbes have been traditionally a diverse and rich source of protease inhibitors and other antibiotics. Since the sequencing of the first Streptomyces genome it has become clear that, at least in the order of Actinomycetales, the genetic capacity for secondary metabolite production exceeds the actually isolated molecules from a certain bacterial strain by large margins. The unknown compounds synthesized by pathways encoded in orphan gene clusters are promising leads in the search for new chemical entities but also for structural analogs of established bioactive substances. An important focus of our research is the discovery of new proteasome and protease inhibitors from bacterial sources. To this end, we are pursuing a targeted genome‐guided approach which takes advantage of the tremendous progress made in the fields of bioinformatics and analytical chemistry. We identified and cloned the gene clusters of two α’,β’‐epoxyketone proteasome inhibitors, epoxomicin and eponemycin. Now, we use the information from our current biosynthetic studies to find orphan gene clusters with analogous organization in public and non‐public data bases. Thereby we focus on genes involved in the formation of pharmacophoric structural features to facilitate the identification of molecules with similar functionality. Subsequently, we make use of our long standing expertise on heterologous systems to express the pathways in surrogate host organisms. LC‐MS‐assisted comparative metabolic profiling is employed to detect the new heterologous compounds. 155 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 127 TTU 9.802 Novel Antiinfectives ‐ DZIF partner site Tübingen, Pharmaceutical Biology Generation of new antibiotics using novel methods of combinatorial biosynthesis and heterologous expression of engineered gene clusters H. Groß1, L. Heide1, B. Gust1, A. K. Apel1 1
Universität Tübingen, Pharmazeutische Biologie, Tübingen, Germany New antibiotics addressing new targets to overcome resistance are urgently needed. Since natural products proved to be extremely successful as antiinfectives, we are dedicated to identify new leads from bacterial producer strains. In order to achieve this goal, we employ new technologies in the field of molecular biology and synthetic biology. These efforts allow us to get access to antibiotics on the one hand and to manipulate the genetic blueprint of antibiotics in order to increase their production or to generate optimized derivatives on the other hand. These techniques were applied to currently underutilized lead compound classes, which address the targets translocase MraY, which is involved in cell wall biosynthesis; the elongation factor Tu (EF‐Tu) which is involved in protein biosynthesis and the B subunit of DNA gyrase. These efforts led within the first funding period to the following new antibiotics: a) two new aminocoumarins, cacibiocin A and B [1,2] were discovered by genome mining b) 11 new uridylpeptides were discovered either by employing combinatorial biosynthesis and synthetic biology or by the application of chemo‐enzymatic methods using an acyltransferase and a sulfotransferase. All derivatives were elucidated and biologically evaluated; as MraY‐inhibitors, most derivatives were highly active towards Mycobacteria and Pseudomonads. c) An engineered Streptomyces coelicolor host strain (M1146) was employed to heterologously express the thiazolylpeptide antibiotic GE2270. A tetracycline inducible promoter (tcp830) was introduced into different positions of the cluster and its influence on GE2270 production was studied [3]. GE2270 A is active against Gram‐positive microorganisms and anaerobes and differs from the other EF‐Tu inhibitors in its spectrum of antimicrobial activity. A derivative has completed clinical phase I/IIa studies as a topical antibiotic against acne [4]. In the upcoming second funding period, we will continue to use the developed tools to find new leads. However, since Gram‐negative pathogens emerge as a growing problem in clinics, particularly the so‐called ESCAPE‐pathogens, we will refocus our program to address these needs, and will search and develop particularly antibiotics which are active towards Pseudomonads, the P of ESCAPE‐pathogens. [1] ChemBioChem (2014), 15(4): 612‐621 [2] Patent application DE 10 2013 021 828.4, 21.12.2013 [3] PLoSONE (2014) 9(3): e90499. [4] Antimicrob Agents Chemother (2015) 59:4560‐4568. 156 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 128 TTU 9 ‐ Novel Antiinfectives German Centre for Infection Research, partner site Tübingen A. K. Apel1, B. Gust1, H. Gross1, L. Kaysser1, L. Heide1, T. Niedermeyer1, Y. Mast1, E. Stegmann1 W. Wohlleben1, N. Ziemert1 1
Eberhard Karls Universität Tübingen, Tübingen, Germany Most antibiotics are natural products or derivatives thereof and are produced as secondary metabolites by bacteria and fungi, likely as warfare agents against competitors. However, due to the rise of multi‐resistant pathogens novel antibiotics and antiinfectives are highly needed. The search for antibiotics with novel targets and the capability to break resistance proved difficult and highly laborious in the past. The partner site Tübingen assembles a number of PI´s comprising all necessary skills for the discovery and the development of novel or improved antibiotics, including modern bioinformatics of genome sequences, heterologous expression in improved host strains, activation of silent gene clusters by regulatory modulation or use of synthetic promoters, combinatorial biosynthesis and synthetic biology approaches. Methods are established for small and large scale fermentation, structure elucidation and biotesting of the novel compounds. Tübingen has a long and successful tradition in secondary metabolite research whose heritage is among others a valuable and still growing strain collection of more than 2000 strains. Sequencing of the genomes of these strains has now started with a first set of 96 actinomycete genomes and will provide Tübingen in time with a priceless database for the discovery of future lead compound candidates. Isolated compounds will be made accessible to all DZIF partner via the DZIF natural compound library. 157 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 129 Evaluation of the in vitro activity of recombinant chimeric bacteriophage endolysin HY‐133 against Staphylococcus aureus small‐colony variants (SCVs) and their corresponding wild types N. Schleimer1, E. Idelevich1, D. Knaack1, G. Peters1, C. von Eiff1, A. Scherzinger2, H. Grallert2, K. Becker1 1
2
Universitätsklinikum Münster, Institut für Medizinische Mikrobiologie, Münster, Germany Hyglos GmbH, Bernried, Germany Introduction Nasal Staphylococcus aureus carriers are of risk to suffer from endogenous infection by this pathogen leading to increased morbidity, mortality and healthcare system burdens. Additionally, small‐colony variants (SCVs) of S. aureus, representing a naturally occurring S. aureus subpopulation frequently isolated in chronic and relapsing infections, hamper the therapy of S. aureus infections. Thus, there is urgent need for alternative prophylactic and therapeutic options. Objectives Previous studies have shown that recombinant chimeric bacteriophage endolysins are highly active against S. aureus and emergence of resistance has not yet been reported. Therefore, this study analyzed the in vitro activity of the endolysin HY‐133 against clinical wild type (WT) isolates and their clonally identical SCVs in comparison to the β‐lactam antibiotic oxacillin. Materials and Methods Bacteriophage endolysin HY‐133 (Hyglos GmbH, Bernried, Germany and oxacillin (Sigma‐Aldrich Co. LLC, Darmstadt, Germany were evaluated regarding their antistaphylococcal activity by broth microdilution method in accordance to CLSI guidelines. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for 12 representative methicillin‐susceptible S. aureus (MSSA) clinical WT isolates, for their corresponding SCVs and S. aureus reference strain ATCC 29213. For all of these isolates, the direct colony suspension method was performed as recommended by CLSI for staphylococci. Furthermore, the activity of HY‐133 and oxacillin was analyzed under exponential growth conditions after 3 h of incubation in liquid medium. Phenotypic stability of the SCVs was controlled throughout the whole experimental process. Results For HY‐133, MBC (mg/L) values were equal to MIC (mg/L) in all isolates. Namely, direct colony suspension method revealed MIC50 and MBC50 values of both 0.12 for WTs and 0.25 for corresponding SCVs. All strain pairs shared the same MIC/MBC90 values of 0.5. For oxacillin, approx. 12.5% of all MBCs were higher than MICs, however, MIC/MBC50 values both were 0.5 for WTs and 0.25 for SCVs and MIC/MBC90 values were found to be 1.0 for all strain pairs. For HY‐133, inoculation of exponential growth cultures revealed MIC/MBC50 values comparable to stationary growth cultures. MIC/MBC90 values were further found to be identical to MIC/MBC90 of direct colony suspension method. Oxacillin also exhibited MIC/MBC50 and MIC/MBC90 values similar to cultures from stationary growth phase. Conclusion This study underlines the high bactericidal activity of HY‐133 against S. aureus WTs and SCVs under different growth conditions. Notably, the bactericidal activity of HY‐133 against the WTs appears to be higher than that of oxacillin. The increase in MBC of oxacillin compared to its MIC was due to strain specific effects and not a general finding. 158 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 130 Significant protection against Staphylococcus aureus targeting novel surface associated proteins by vaccination with humanized antibodies or their corresponding epitope‐
peptides A. Klimka1, A. Nicolai1, S. Mertins1, F. Nickel1, O. Krut1, M. Krönke1 1
Uniklinik Köln, Inst. Med. Mikrobiologie, Cologne, Germany
The fact that an increasing number of patients infected with antibiotic‐resistant, life‐threatening bacteria can be treated only, if at all, with a few, not well tolerated antibiotics of last resort, calls for new and strong efforts on this matter. An alternative to bactericidal or bacteriostatic compounds are vaccination strategies, triggering or supporting the patients’ immune system to cope with the deadly infection. Concerning multi‐resistant Staphylococcus aureus, many research groups and pharmaceutical companies have developed vaccines, targeting numerous, well known virulence factors of this major nosocomial pathogen but none of them reached the market so far. We identified three surface‐associated proteins of S. aureus as vaccine candidates, using patient‐derived immunoglobulins for subtractive proteome analysis: Hp2160 is a hypothetical protein of unknown function with an esterase‐like domain. Protoporphyrinogen oxidase (pOxi) is involved in heme‐synthesis and triose phosphate isomerase (Triiso) is a glycolytic enzyme. These proteins have additional `moonlight´ activities on the bacterial surface, as adhesins, invasins or plasminogen receptor and are so far unrecognized, highly conserved, pathogenicity factors demonstrating significant protective effects in a S. aureus infection mouse model. Furthermore, we generated murine monoclonal antibodies (moAbs) against each of these target proteins, of which three demonstrated significant, epitope‐specific protection against S. aureus infection. Although the mode of action of these moAbs against S. aureus will be investigated in greater detail together with our DZIF cooperation partners, they seem not to enhance phagocytosis, yet interfere with the S. aureus‐host interaction, specifically with heme‐sensing, ‐uptake and plasminogen binding. These moAbs retained their protective effect after been humanized and de‐immunized, and will be used in First‐In‐Man studies. So far, the selected anti‐pOxi humAb‐producing cell line is in the process of pre‐GMP‐production to retrieve GMP‐like humAb for the necessary pre‐clinical studies. We have identified the epitopes of these moAbs to be used as combined, multivalent peptide vaccine against S. aureus infection. Immunization with the epitope‐peptides or a single‐stranded tri‐epitope‐peptide leads to antigen‐specific IgG titers, providing a significant bacterial clearance in examined organs in our sublethal S. aureus infection model. We have improved previous anti‐S. aureus vaccination approaches by distinguishing protective from non‐
protective epitopes of selected novel target antigens. Passive vaccination will be of valuable means to protect against S. aureus, where antibiotics fail. Epitope‐peptide vaccination will provide a safe, inexpensive and efficient measure for patients confronted with a high‐risk of S. aureus infection. 159 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 131 ADMET of corallopyronin A, a novel antibiotic S. Bouhired1, A. Schiefer1, K. Pfarr1, G. M. König2, A. Hörauf1 1
2
Universitätsklinikum Bonn, Institut für Medizinische Mikrobiologie, Immunologie und Parasitologie, Bonn, Germany Universität Bonn, Institut für Pharmazeutische Biologie, Bonn, Germany The natural compound corallopyronin A, isolated from the myxobacterial strain Corallococcus coralloides B035, showed antibiotic activity against the endobacteria Wolbachia, where it inhibits the bacterial DNA dependent RNA polymerase. In contrast to rifampicin, it has a new mode of action* and is thus not only active against Gram‐positive bacteria, but also against rifampicin‐resistant S. aureus**. For the development of a pharmaceutical drug, physicochemical properties and pharmacokinetics have to be characterized. Drug solubility is important for dose regimen and biotechnological production. Corallopyronin A is a lipophilic, weakly acidic, amorphous compound with good solubility in ethanol, methanol and DMSO, but poor solubility in water. To improve the solubility in water several common counter‐ions and solutions have been tested to achieve a crystalline form of the compound, but only amorphous forms of corallopyronin A and its salts were obtained. Nevertheless, despite the poor solubility, there are no issues with the oral bioavailability. Our studies have also shown that with increased pH values the compound is better soluble and that the best solubility is reached at blood pH (7.4). Due to its lipophilic character, it had better solubility in FESSIF medium. Corallopyronin A shows a high plasma protein binding, which is comparable to warfarin and glimepirid. Regarding drug‐drug interaction of the compound, our studies showed that it is a weak inducer of CYP3A4 in comparison to rifampicin and therefore should not affect the metabolism of other antibiotics/therapeutics. These results highlight areas requiring greater focus for the further development of corallopyronin A as an antibiotic, especially for the formulation of a Galenic.Thus, current efforts focus on making solid corallopyronin A by using other counter‐ions and solutions as well as alternative drying methods after purification from the fermentation culture. *
Schiefer et al. “Corallopyronin A Specifically Targets and Depletes Essential Obligate Wolbachia Endobacteria From Filarial Nematodes In Vivo.” The Journal of Infectious Diseases 206.2 (2012): 249‐257 **
Erol et al.“ Biosynthesis of the myxobacterial antibiotic corallopyronin A.“ ChemBioChem (2010) 11(9):1253‐
65 Figure 1 160 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 132 Labyrinthopeptins ‐ New Lantibiotics with broad anti‐viral activity H.‐ P. Prochnow1, S. Gordts2, S. Haid3, S. Blockus3, T. Schulz4, T. Pietschmann3, D. Schols2, M. Brönstrup1 1
Helmholtz Zentrum für Infektionsforschung, Chemische Biologie, Braunschweig, Germany Universität, Leuven, Belgium 3
Twincore, Hannover, Germany 4
Medizinische Hochschule, Hannover, Germany 2
Labyrinthopeptins A1 and A2 (LabyA1, LabyA2) are modified peptides produced by the desert bacterium Actinomadura namibiensis. They are the first congeners of a novel class of lantibiotics, characterized by the presence of carbacycles spanned by the amino acid labionin with its αC‐quaternary substitution (Fig. 1). Following a first report on the broad anti‐HIV and anti‐HSV activities of LabyA1 (Férir et al. 2013), we have extended our studies to other viruses such as the Dengue virus (DENV). The anti‐DENV activity of Laby was determined through automated fluorescence microscopy following DENV infection of the human hepatocyte cell line Huh‐7. Our data indicate that LabyA1 is a more potent inhibitor of viral infection (IC50 = 3.7 µg/ml, Fig. 2) than LabyA2 (IC50 = 15.4 µg/ml). LabyA1 also dose‐dependently inhibited the replication of the four serotypes of DENV in DC‐SIGN‐transfected Raji cells and in monocyte‐derived dendritic cells (MDDC) with IC50’s of 0.6‐3.2 μg/ml. Time‐of‐drug‐addition experiments indicated that Laby impairs viral entry through binding to the virus itself rather than binding to the host cell or acting after the virus has entered the cell. Furthermore LabyA1 inhibited DENV‐induced fusion of insect cells, suggesting an interaction with the fusion region of the viral E‐glycoprotein. We are currently probing proteinaceous interaction partners of LabyA1 and LabyA2 on the viral envelope through peptide microarray experiments. Fig.1: Structures of LabyA1 and LabyA2. Lab = Labionin; Dhb = Dehydrobutyrate Fig. 2: Anti‐Dengue‐viral activity of LabyA1. The percentage of Dengue‐infected cells 48h post‐infection is expressed as “% Alexa488 positve” (left axis). The logIC50 is 0.57, corresponding to 3.7 µg/ml. As indicated by the total number of cells visualized by DAPI‐staining (right axis), LabyA1 shows no toxic effects up to 50 µg/ml. Figure 1 Figure 2 161 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 133 Cationic amphiphilic drugs enhance entry of lentiviral particles pseudotyped with rabies virus glycoprotein into non‐neuronal cells A. Klintworth1, T. Nolden2, S. Westhaus1, K. Rohrmann1, S. David3, M. Manns4, S. Finke2, S. Ciesek4, T. von Hahn1 1
Medizinische Hochschule Hannover, Molekularbiologie/ Gastroenterologie, Hannover, Germany Friedrich‐Loeffler‐Institut, Institut für molekulare Virologie und Zellbiologie, Greifswald ‐ Insel Riems, Germany 3
Medizinische Hochschule Hannover, Nephrologie, Hannover, Germany 4
Medizinische Hochschule Hannover, Gastroenterologie, Hannover, Germany 2
Cell entry mediated by the rabies virus (RABV) glycoprotein (GP) involves interaction with cell surface molecules followed by clathrin‐mediated endocytosis. Amiodarone and other cationic amphiphilic drugs (CADs) inhibit cell entry by diverse human pathogenic viruses including Filoviruses, Dengue virus and Japanese encephalitis virus. They are thus considered potential broad spectrum antiviral agents. In study we examined the effect of amiodarone and other CADs on RABV glycoprotein‐ (GP‐) mediated cell entry of pseudotyped lentiviruses. Here we report the unexpected finding that amiodarone and other CADs markedly enhance RABV glycoprotein‐ (GP‐) mediated cell entry of pseudotyped lentiviruses into non‐neuronal cells but not in neuronal cells. Increased cell entry seems to be due to effects of the drugs on the host cell rather than the pseudoviral particles and can also be elicited when CADs are added several hours after pseudoviral attachment. There is no detectable CAD‐induced change in the expression of putative RABV cell surface receptors. Perturbing endosomal processing with phosphoinosite‐3‐kinase inhibitors wortmannin and LY294002 mimics the effects of CADs on RABV GP‐mediated cell entry. Thus, CADs may enhance RABV GP‐mediated cell entry of pseudotyped lentiviruses by promoting a late step of the pseudoviral cell entry process, possibly release from an endosomal compartment into the cytosol. In contrast to the pseudotyped lentiviruses, infection by fully infectious RABV was not enhanced by CADs, indicating, that the observed stimulation of RABV GP mediated lentivirus entry also depended on the used lentivirus vector backbone. In conclusion, we show that while CADs inhibit cell entry of diverse viruses they can also have a paradoxical enhancing effect on the ability of a viral glycoprotein to mediate cell entry depending on the cellular and viral context. This needs to be taken into account when considering use of CADs as antiviral agents. 162 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship
P 134
Preclinical Discovery and development of antivirals from natural products
R. Brack-Werner1, S. Rebensburg1, M. Helfer1, M. Schneider1, H. Koppensteiner1, M. Schindler2, L. Gürtler3
1
Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt, Institut für Virologie,
Oberschleißheim, Germany
2
University Hospital of Tübingen, Department of Medical Virology and Epidemiology of Viral Diseases, Tübingen, Germany
3
Ludwig Maximilian's University, Max von Pettenkofer Institute, Munich, Germany
There is a strong need for novel antiviral agents for the treatment of life-threatening viral infections such as HIV
and Ebolavirus. We aim to develop antivirals that display modes-of-action different from approved drugs.
Therefore we developed screening technology that covers the entire HIV replication cycle and allows unbiased
identification of HIV inhibitors (EASY-HIT). This technology is based on HIV-susceptible human cells with an
integrated reporter gene that requires HIV Rev and Tat proteins for expression. Infection levels in virusexposed cultures are quantified by measuring the red fluorescent reporter signal. The EASY-HIT technology is
not limited to HIV and can be used in combination with pseudotyped virus particles for identification of entry
inhibitors against numerous different viruses. We used the EASY-HIT system to screen over 25000 single
compounds and compound mixtures of varying complexities. Interestingly, the most promising hits in terms of
antiviral potency and novelty of inhibitory activity turned out to be natural products. Our current efforts are
focusing on preclinical studies of compound classes that inhibit virus entry or virus expression by new modesof-action. Natural products have contributed to the development of a substantial proportion of currently
approved medical drugs, based on their high chemical diversity and multiple bioactivities. Our studies support
the use of natural products as a source for the development of novel antiviral drugs.
Albert Einstein, verwendet mit
Erlaubnis von HUJ / GreenLight
BE THE
ONE
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EINFACHE FORMEL BRINGT
HARVONI® – 1 TABLETTE / TAG a, d
Heilung bei bis zu 99 % 1–4 aller HCV GT 1-Patienten
Einfach für Arzt und Patient – 99 % Compliancee
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94 % – 99 % Heilungsrate (SVR12) bei
therapienaiven Patienten in 8 b– 24 Wochen (ION-3, ION-1)
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Frei von Interferon , Ribavirin d und
Proteaseinhibitoren
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86 % –100 % Heilungsrate (SVR12) bei
vorbehandelten Patienten in 12 – 24 c Wochen (ION-2)
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Option auf kurze 8 b-Wochen-Therapie
bei therapienaiven Patienten ohne Zirrhose
b
a HARVONI ® ist zugelassen zur Behandlung der chronischen Hepatitis C bei Erwachsenen, Fachinformation HARVONI ®, Stand Juni 2015 . b Empfohlene Therapiedauer für therapienaive Patienten ohne Zirrhose : 12 Wochen ; eine 8-Wochen-Therapie ist bei Patienten
mit einer HCV-RNA-Viruslast < 6 Mio. IU/ml in Erwägung zu ziehen . c Es ist zu erwägen ,
vorbehandelte Patienten ohne Zirrhose mit ungewissen nachfolgenden Wiederbehandlungsoptionen 24 Wochen zu behandeln . d Ausnahme: Patienten mit dekompensierter
Zirrhose sowie pre- und post-transplant-Patienten erfordern die zusätzliche Gabe
von RBV. e In klinischen Studien der Phase III unter einer 12-Wochen-Therapie , ION-1 –3.
1 Afdhal N et al. N Engl J Med. 2014; 370: 1889–1898. (ION-1) 2 Afdhal N et al. N Engl J Med.
2014; 370: 1483–1493. (ION-2) 3 Kowdley KV et al . N Engl J Med . 2014 ; 370 : 1879 –1888 .
(ION-3) 4 Fachinformation HARVONI ®, Stand Juni 2015.
3350, Talkum, Gelborange-S-Aluminiumsalz (E110). Anwendungsgebiet : HARVONI ® wird zur
Behandlung der chronischen Hepatitis C (CHC) bei Erwachsenen angewendet (siehe Abschnitte 4.2, 4.4 und 5.1 der Fachinformation).Gegenanzeigen : Überempfindlichkeit gegen
die Wirkstoffe oder einen der sonstigen Bestandteile. Gleichzeitige Anwendung mit Rosuvastatin oder Johanniskraut (Hypericum perforatum). Nebenwirkungen : Sehr häufig
( • 1 / 10 ): Kopfschmerzen , Erschöpfung . Beschreibung ausgewählter Nebenwirkungen:
Herzrhythmusstörungen. Fälle von schwerer Bradykardie und Herzblock wurden bei der Anwendung von HARVONI® bei gleichzeitiger Anwendung von Amiodaron und/oder Arzneimitteln, die die Herzfrequenz senken, beobachtet (siehe Abschnitte 4.4 und 4.5 der Fachinformation). Darreichungsform und Packungsgrößen: Packung mit 28 Filmtabletten.
Verschreibungspflichtig. Stand: Juni 2015. Pharmazeutischer Unternehmer: Gilead Sciences
International Ltd., Cambridge, CB21 6GT, Vereinigtes Königreich. Repräsentant in Deutschland: GILEAD Sciences GmbH, D-82152 Martinsried b. München
HARVONI® 90 mg / 400 mg Filmtabletten
Wirkstoffe : Ledipasvir und Sofosbuvir . Zusammensetzung : Jede Filmtablette enthält 90 mg
Ledipasvir und 400 mgSofosbuvir. Sonstige Bestandteile: Tablettenkern: Copovidon, Lactose-Monohydrat, Mikrokristalline Cellulose, Croscarmellose-Natrium, Hochdisperses Siliciumdioxid, Magnesiumstearat (Ph.Eur.). Filmüberzug: Poly(vinylalkohol), Titandioxid, Macrogol
Dieses Arzneimittel unterliegt einer zusätzlichen Überwachung. Jeder Verdachtsfall einer
Nebenwirkung zu HARVONI® ist zu melden an die Gilead Sciences GmbH, Abteilung Arzneimittelsicherheit, Fax-Nr.: 089/899890-96, E-Mail: [email protected], und/oder
an das Bundesinstitut für Arzneimittel und Medizinprodukte, Abt. Pharmakovigilanz, KurtGeorg-Kiesinger Allee 3, D-53175 Bonn, Webseite: www.bfarm.de.
Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 135 Cellular pathways involving cytosolic phospholipase A2a may represent an antiviral drug target suitable to combat infections caused by newly emerging coronaviruses and other plus‐strand RNA viruses C. Müller1, S. Pleschka1, J. Ziebuhr1 1
JLU Giessen, Medizinische Virologie, Giessen, Germany To combat infections caused by newly emerging RNA viruses more efficiently, new broad‐spectrum antivirals that either target cellular pathways essential to viral replication or viral structures/mechanisms that are conserved across whole virus families would be highly desirable. In this study, we investigated antiviral effects of a non‐peptidic, low‐molecular‐weight inhibitor of the cytosolic phospholipase A2a (cPLA2a) against coronaviruses (HCoV‐229E, MERS‐CoV). The inhibitor was demonstrated previously to be active against Dengue virus and HCV (Menzel et al., 2012). We found that non‐cytotoxic concentrations of the inhibitor (Py‐2) exhibited profound effects on (i) the production of infectious virus progeny, (ii) viral protein accumulation and (iii) viral RNA synthesis in Huh7 cells. The significance of cPLA2a activity in coronavirus replication was further supported by data showing that inhibitors of upstream activator kinases of cPLA2a (MEK and p38) affect coronavirus replication. Furthermore, in the presence of the inhibitor, coronaviral dsRNA intermediates as well as virally encoded subunits of the replication/transcription complex (RTC) could not be detected, suggesting that inhibition of cPLA2a activity interferes with the formation of active RTCs at virus‐induced intracellular double‐membrane structures. Of note, influenza virus replication was not affected by the cPLA2a inhibitor. Combined with previous studies on hepatitis C and dengue viruses, our data identify cPLA2a as an important cellular activity involved in the replication of diverse plusstrand RNA virus families and suggest that specific inhibitors of cPLA2a are promising drug candidates for combating infections caused by newly emerging plus‐
strand RNA viruses. 164 Antibiotic resistant bacterial infections – Novel antiinfectives and antibiotic stewardship P 136 Burden and Spectrum of Infectious Disease in Germany 2009‐2014: A Multicentre Study from Berlin’s Municipal Hospitals J. Katchanov1, D. Tominski1, L. Jeffreys1, H. Slevogt2, K. Arasteh1, H. Stocker1 1
2
Auguste Viktoria Klinikum, Infectious Diseases, Berlin, Germany Universität, Jena, Germany Purpose To assess theburdenand spectrum of infectious diseases (ID) in a Metropolitan population in Germany. Methods A discharge database using ICD‐10 codes enabled the identification of hospitalizations with infection‐related diagnoses. All hospital admissions between 2009 and 2014 were analysed from 9 municipal hospitals serving approximately one third of an urban population of 3.5 million people. Results We identified114,168 admissions with a primary (first‐listed) ID diagnosis and 220,483 admissions with any‐
listed ID diagnosis, accounting for 8.9% (95% confidence intervall (CI): 8.9%‐9.0%) and 17.2% (95%CI: 17.1‐17.3) of all 1,284,559 admissions, respectively. Annually, 439,837 bed‐days (range 413,707‐ 488,520) were occupied by patients with an ID diagnosis, utilizing 22.8% of total bed capacity.The median length of stay for patients with primary ID diagnosis and secondary ID diagnosis was 6 days (IQR 3‐11) and 10 days (IQR 5‐19), respectively.The most common diagnosis across all age groups was “pneumonia” (22.8% and 16.2% of ID admissions as primary and secondary diagnosis, respectively). In‐hospitalmortality was 6.8% (95%CI 6.6‐6.9) and 8.9% (95%CI 8.7‐9.1) for ID as primary and secondary diagnosis, respectively. Conclusion Infectious diseases contribute significantly to the overall burden of disease in a health system caring for an urban German population. In view of the magnitude of ID’s contribution, establishing more specialists in ID medicine and adjusting the reimbursements for managing infection‐related admissions should be made a public health priority in Germany. 165 Chronic Viral Infections: HIV and Hepatitis P 137 Bispecific antibody constructs mediate specific retargeting towards and elimination of HBV‐positive hepatocytes and tumor cells F. Bohne1, J. Hasreiter1, O. Quitt1, J.‐ H. Bockmann1, F. Momburg1, U. Protzer1 1
Helmholtzzentrum München, Institut für Virologie, Munich, Germany Background and Aim Chronic hepatitis B is characterized by dysfunctional effector cells, incapable of controlling the virus. HBV‐
specific retargeting of immune effector cells using bispecific monoclonal antibody (BiMab) constructs is a promising therapeutic approach to circumvent T‐cell dysfunction. In this study, effector cells are supplied with BiMab that redirect T cells towards HBV‐infected hepatocytes resulting in cytotoxic elimination. Methods We developed tetravalent BiMab harboring four single chain fragment (scFv) binding domains. The first scFv targets the small HBV‐envelope protein (HBs) on the plasma membrane of infected hepatocytes. The second binding motif engages T cells either via CD3 or co‐stimulates them via CD28. The two binding moieties are connected through an IgG1‐derived Fc‐domain. Two such constructs homodimerise to form the tetravalent BiMab suitable to interconnect the target and the effector cell resulting in recruitment and activation. Results In co‐culture experiments, the BiMab‐mediated redirection of immune effector cells towards HBs‐positive hepatoma cells induced specific elimination of target cells and activation of effector cells. Co‐administration of HBs/CD3‐ and HBs/CD28‐specific BiMab constructs resulted in specific and synergistic elimination of up to 96% of HBs‐positive target cells in co‐cultures with PBMC. Intracellular cytokine staining showed polyfunctional activation of T cells mediated by the BiMab. In both, CD4 and CD8 positive T cells, simultaneous secretion of IFNγ, IL‐2 and TNFα implicated a fully functional redirection. Importantly, BiMab were also able to redirect T cells to HBV‐infected HepaRG cell resulting in specific killing. Furthermore, soluble HBs, mimicking the high viral loads in patient serum failed to induce effector cell activation even when added to non‐infected cells. Finally, first in vivo experiments in immunedeficient mice transplanted with HBV‐positive hepatoma cells to induce subcutaneous tumors showed that transferred human PBMC mediated a marked decrease in tumor size upon BiMab injection indicating a functional homing into tumors. Conclusion Retargeting of T cells towards HBV‐positive target cells using bispecific antibody constructs is a promising new immunotherapeutic approach to treat chronic hepatitis B and associated hepatocellular carcinoma. 166 Chronic Viral Infections: HIV and Hepatitis P 138 Chronic pathogen‐induced liver inflammation: Interaction of Hepatitis B virus infection and Schistosoma in a mouse model E. Loffredo‐Verde1, C. Prazeres da Costa1, U. Protzer1, A. Malo1, D. Busch1 1
Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Mikrobiologie, Munich, Germany One of the main complications of acute HBV infection is the development of a chronic infection. Interestingly, chronicity develops at a higher frequency in developing countries in which co‐infections with several helminth species, such as S. mansoni, are common. The underlying mechanisms leading to HBV chronicity are only partly understood but compromise CD8+ and CD4+ T cell responses, which might be affected in helminth co‐infected patients due to dynamic schistosome‐driven immune responses. These range from an initial Th1‐ to Th2‐
immunity and eventually to long‐term immunosuppression. Thus, the effects of pre‐existing helminth infections on concomitant HBV infection are hard to predict and need clarification. We investigated the impact of acute and chronic schistosomiasis on the outcome of an additionally acquired, acute HBV infection in mice. As a result, acute HBV infection acquired during the Th1 phase of schistosomiasis was suppressed in co‐infected animals when compared to HBV mono‐infected mice, since HBsAg and HBeAg were significantly reduced in co‐infected animals. Additionally, less HBsAg‐ and HBcAg‐positive hepatocytes were detected in liver sections in co‐infected mice, indicating that viral elimination occurs at a very early timepoint. Furthermore, co‐infected animals showed higher frequencies of CD4+ IFNγ+ and CD8+ IFNγ+ T cells after antigen‐specific (HBcP3) and antigen‐unspecific (PMA/Ionomycin) stimulation. Interestingly, acute HBV infection acquired during the chronic phase of S. mansoni infection was also cleared faster in co‐infected mice when compared to HBV mono‐infected mice, since these animals showed again decreased viral antigens and increased virus‐specific immune responses. Taken together, our data indicate a causal relationship between schistosome‐induced IFN‐γ production within the liver, the induction of antiviral T cells and clearance of HB Virus. 167 Chronic Viral Infections: HIV and Hepatitis P 139 Investigation of resistance‐associated variants for optimization of direct antiviral therapy of hepatitis C virus genotype 1‐infected patients J. Dietz1, S. Susser1, C. Berkowski1, D. Perner1, S. Passmann1, S. Zeuzem1, C. Sarrazin1 1
University Hospital Frankfurt, Medical Department 1, Frankfurt, Germany Background Currently different highly effective interferon‐free treatment options for patients with chronic hepatitis C virus (HCV) infection are available. These include combinations of a nucleotide NS5B inhibitor (sofosbuvir, SOF) with a NS3 protease inhibitor (simeprevir, SMV) or a NS5A inhibitor (Daclatasvir, DCV or Ledipasvir, LDV). An alternative option is a triple DAA (direct‐acting antiviral agents) therapy (Paritaprevir, PTV; Ombitasvir, OMV; Dasabuvir, DSV) for genotype (GT) 1. In the respective studies, sustained virologic response rates (SVR) were high (up to 99%) but pre‐existent resistance‐associated variants (RAVs) reduced SVR rates of a range between 5 and 50%. Methods Serum of 512 European patients with a chronic GT1 HCV infection was collected and used for the parallel amplification and population‐based sequencing of NS3, NS5A and NS5B genes. 409 patients were treatment‐ and DAA‐naïve and 103 individuals were treatment‐experienced. Pretreatment occurred in 65% with pegylated interferon/ribavirin, in 34% with a protease inhibitor (here telaprevir/boceprevir were mainly administered, 77%) and 1% received ledipasvir/tegobuvir. NS3 RAVs with relevance for SMV/PTV were analyzed at positions F43, Y56, Q80, R155, D168 and investigation of NS5A RAVs for DCV, LDV and OMV was performed at positions M28, Q/R30, L31, P32, H/P58 and Y93. Non‐nucleoside inhibitor NS5B RAVs for DSV were examined at positions C316, S368, Y448, S556, and D559. Results We detected RAVs with important differences for HCV subtypes. NS3 RAVs were found in 21% of treatment‐
naïve and 16% of ‐experienced individuals. Although pretreatment was also performed using protease inhibitors, the number of NS3 RAVs was comparable in both groups and Q80K (36%) and D168 variants (2%) were the most frequent RAVs in GT1a and 1b respectively. Within NS5A we identified RAVs in 13% of treatment‐naïve and 22% of ‐experienced patients and most common were M28V and Q30 variants (6%) in GT1a and Y93H (12%) in GT1b. The prevalence of NS5B RAVs was 24% in treatment‐naïve and 30% in ‐
experienced individuals. Here, GT1a RAVs occurred rarely with a frequency of below 4% (mainly S556 variants), whereas RAVs were detected in nearly one half (42%) of 1b patients (predominantly C316 variants and S556G). However, no marked differences in the prevalence of RAVs between treatment‐naïve and ‐experienced patients were detected. Under consideration of all three DAA targets, 99% of patients with subtype1a and 98% of patients with subtype 1b had no RAVs for at least one currently available interferon‐free DAA regime. Conclusions The testing of baseline resistance allows the selection of at least one RAVs‐free treatment option for nearly all treatment‐naïve and ‐experienced patients which enables a potentially cost‐optimized chronic hepatitis C treatment. 168 Chronic Viral Infections: HIV and Hepatitis P 140 Multiplex serology for Hepatitis A and Hepatitis E Virus Infections K. Bohm1, G. Krause1, C. Sievers1 1
Helmholtz‐Zentrum für Infektionsforschung, Braunschweig, Deutschland, Epidemiologie, Braunschweig, Germany Introduction Infections with hepatitis A virus (HAV) and hepatitis E virus (HEV) both can result in acute viral hepatitis. Existing assays for serological trials of hepatitis virus infections can only measure the presence of one type of antibody. An assay for screening antibodies against different hepatitis virus infections at once, at low cost and requiring a small amount of serum, can be a useful method not only for the individual diagnosis of hepatitis virus infections but also in population based sero‐surveys. Objectives The aim of this project is to establish a serological tool called multiplex serology for the detection of antibodies specific for HAV and HEV infections with the secondary aim to distinguish between natural (due to infection) and vaccine‐induced immune responses. Materials and Methods The system of multiplex serology is based on the technique to conjugate recombinant antigens to color coded beads which are then used to catch their specific antibody from serum. Using a dual laser system (Luminex®), the bead type and fluorescence intensity of the bound antibodies are detected. Unlike ELISA, multiplex serology allows the distinctive analysis of a large number of sera for antibodies against many different antigens at the same time, utilizing less than 10 µl serum. Results Using multiplex serology with 6 structural and nonstructural HAV antigens, we detected naturally infected HAV‐
IgG positive serum samples with a sensitivity of 95% and specificity of 97.4%. The HAV serum samples of previously HAV‐vaccinated individuals were positive in a standard hepatitis A ELISA. Using a combination of HAV multiplex serology and standard HAV ELISA for a subset of both, previously HAV‐vaccinated and previously HAV‐infected samples, we identified previously infected subjects with a sensitivity of 92.1% and specificity of 98.3%. HEV‐IgG‐positive serum samples were detectable using multiplex serology with a sensitivity of 100% and specificity of 94%. Conclusion To serologically distinguish between previously HAV‐infected and HAV‐vaccinated samples, the combination of two different laboratory methods are currently used. We are in process to extend the antigen pool to include conformational antigens for HAV to allow a more rapid differentiation between vaccinated and naturally infected samples, i.e. in the multiplex approach. Regarding HEV multiplex serology, one antigen suffices to differentiate between HEV‐IgG negative and HEV‐IgG positive serum samples. Distinction between previously vaccinated and naturally infected individuals can provide information to inform vaccination strategies, also with regard to recently made available HEV vaccine. Subject to further validation, our approach could provide a valuable tool for efficient population based sero‐surveys, for individual differential diagnosis, for studies on vaccination coverage and for outbreak investigations. 169 Chronic Viral Infections: HIV and Hepatitis P 141 HCV‐specific immune responses under direct‐acting antiviral therapy of chronic HCV infection C. Dembek1,2,3, C. Russo1, A. Grambihler4, J. Schattenberg4, T. Zimmermann4, A. Weinmann4,5, P. Galle4 U. Protzer1,2,3,6, T. Bauer1,2,3, M. Sprinzl4,5 1
Helmholtz Zentrum München, Institute of Virology, Munich, Germany Helmholtz Zentrum München, Cooperation Group‐ Immune Monitoring, Munich, Germany 3
German Center for Infection Research, Munich, Germany 4
University Medical Center, I. Medical Department, Mainz, Germany 5
University Medical Center, Clinical Registry Unit, Mainz, Germany 6
Institute of Virology, Technische Universität München, Munich, Germany 2
Background Direct‐acting antiviral drugs (DAA) with or without Ribavirin or PEG‐Interferon‐alpha efficiently suppress and clear hepatitis C virus (HCV) infection. However, it remains controversial if robust HCV suppression also affects viral immune‐escape and restores HCV‐directed adaptive immune responses in vivo. Methods In order to address these issues we monitored HCV‐specific T‐cell response from 20 chronically HCV infected patients throughout treatment with Sofosbuvir, Ribavirin and PEG‐Interferon‐alpha. HCV‐specific CD4 and CD8 T‐cell responses were identified by ex vivo HCV epitope pool stimulation and simultaneous detection of 6 functional markers. Results The sustained virological response (SVR) rate was 95 percent, in the study cohort of HCV Genotype 1a/b (n=7/13) infected patients including cirrhotics (n=4/16). First results from 10 of 20 treated patients indicate re‐
activation of HCV‐specific CD4 T‐cells characterized by TNF‐α and IL‐2 production at 12 weeks after the end of treatment. However, total cytokine expression by NS3‐specific CD8 T‐cells was significantly reduced at 12 weeks after the end of treatment compared to therapy initiation. Discussion These data suggest a re‐constitution of HCV‐specific CD4 T‐cells but not of CD8 T‐cells following Sofosbuvir, Ribavirin and PEG‐Interferon‐alpha combination therapy. Further investigations are ongoing to identify HCV‐ responses during IFN‐free DAA regimens, to confirm the HCV‐specific T‐cell response pattern without therapeutic immune stimulation. 170 Chronic Viral Infections: HIV and Hepatitis P 142 The course of Hepatitis E virus genotype 3 infection in wild boar and pigs: Immune response and transmission characteristics in chronically and experimentally infected animals M. Eiden1, J. Schlosser1, L. Dähnert1, H. Sheikh Ali1, U. Blohm2, A. Vina‐Rodriguez1, C. Fast1, R. G. Ulrich1 M. H. Groschup1 1
2
Friedrich‐Loeffler‐Institut, Institut für Neue und Neuartige Tierseuchenerreger, Greifswald‐Insel Riems, Germany Friedrich‐Loeffler‐Institut, Institut für Immunologie, Greifswald‐Insel Riems, Germany Hepatitis E virus (HEV) is a small, non‐enveloped virus whose genome is formed by a single RNA strand with positive polarity and a length of approximately 7.3 kbp encoding three open reading frames (ORF1‐3). Hepatitis E virus (HEV) is an emerging pathogen and a major causative of acute viral hepatitis in humans, primarily in developing countries. Within the genus Orthohepevirus genotypes 1 and 2 (HEV‐1, ‐2) are exclusively detected in humans, whereas genotypes 3 and 4 (HEV‐3, ‐4) are zoonotic viruses with porcine species being the primary reservoir hosts. An increasing number of autochthonous cases of Hepatitis E have been reported in industrialized countries, which are linked to the consumption of contaminated meat and blood transfusion. HEV infections in humans can cause chronic hepatitis especially in immuno‐compromised patients which can lead to progressive cirrhosis eventually. In analogous manner HEV infections in porcine species can cause a persistent viraemia. Here we describe the experimental infection of wild boar and domestic swine as animal models for the human HEV infection. For the first time we were able to follow up the HEV infection in formalin‐fixed, paraffin‐
embedded tissues by immunohistochemical (IHC) staining using polyclonal antibodies raised to recombinant HEV capsid protein. HEV antigen was consistently detected in liver from all i.v.‐infected animals and Kupffer cells exhibited the strongest IHC signals. Viral antigens were also found in spleen and in hepatic, mandibular and mesenteric lymph nodes. In addition we investigated the characteristics of Hepatitis E in chronically infected wild boar which was still transmissible to domestic pigs. 171 Chronic Viral Infections: HIV and Hepatitis P 143 Peak of HBV‐DNA after stopping NA therapy in HBeAg‐negative chronic patients is associated with T cell responses and subsequent HBsAg decline F. Rinker1,2, C. Höner zu Siederdissen 1, C. M. Bremer3,4, B. Bremer1, C. S. Falk5, M. Manns1,2 H. Wedemeyer1,2, D. Glebe3,4, A. R. Kraft1, M. Cornberg1,2 1
Hannover Medical School, Gastroenterology Hepatology and Endocrinology, Hannover, Germany German Center for Infection Research (DZIF), Partner Side HepNet Study‐House, Hannover, Germany 3
Institute of Medical Virology, Justus Liebig University Giessen, National Reference Center for Hepatitis B and D Viruses, Giessen, Germany 4
German Center for Infection Research (DZIF), Partner Side Giessen, Giessen, Germany 5
Hannover Medical School, Institute of Transplant Immunology, IFB‐Tx, Hannover, Germany 2
Introduction Stopping nucleos(t)ide analogue (NA) treatment before HBsAg loss is currently debated as a concept to achieve HBsAg loss. In a prospective single center study we show that stopping NA treatment in HBeAg‐negative patients is safe and leads to a significant decline of HBsAg. To understand mechanisms leading to HBsAg decline, the three HBV surface proteins (S‐, L‐ and MHBs), HBV core‐related antigen (HBcrAg), cytokine/chemokine levels and HBV‐specific T cell responses were measured before and after stop of NA therapy. Methods In a prospective pilot trail, NA treatment was stopped in 15 chronically hepatitis B infected HBeAg‐negative patients. Patients with ongoing NA therapy, suppressed HBV DNA (<20 IU/ml) for at least three years and HBsAg levels less than 3500 IU/ml were included in this study. When a virological relapse occurred (HBV DNA >2000 IU/ml) patients were retreated. With an in‐house ELISA we analyzed the amount of the three HBs proteins in the sera of patients. Serum cytokine/chemokine profiles were measured using multiplex technology (Bio‐Plex System). HBV‐specific T cell responses were determined by an intracellular cytokine staining after stimulating PBMCs with HBV core‐specific overlapping peptides. Results In 13/15 patients a virological relapse with HBV‐DNA and ALT flairs at week 4 (n=4), 8 (n=11), 24 (n=1) and week 36 (n=1) was detected. On the last observation two patients lost HBsAg, with one having anti‐HBs. One additional patient almost lost HBsAg (0.2 IU/ml).The peak of HBV‐DNA and HBcrAg during relapse correlated strongly with fold decline of HBsAg at week 48. There was not such a correlation with ALT. Overall levels of S‐, L‐ and MHBs significantly declined as seen for qHBsAg. Plasma cytokines/chemokine levels of IL‐10, IL‐12 and TNF‐α were significantly increased early (4 weeks) and CXCL10 (IP‐10) at week 8 after treatment stop. Interestingly, some but not all patients showed increased functional HBV‐specific T cell responses upon stop of NA treatment. Conclusion In summary, stopping NA therapy leads to a significant HBsAg decline strongest in patients with highest HBV‐
DNA and HBcrAg levels. Our data suggest that cytotoxic and non‐cytotoxic immune responses might be involved and that the right time point of NA retreatment is absolutely important. 172 Chronic Viral Infections: HIV and Hepatitis P 144 Geno2pheno[HCV] ‐ A Web‐Service to Support Hepatitis C Treatment Decisions in the Era of Direct‐Acting Antiviral Agents P. Kalaghatgi1, B. Beggel1, E. Knops2, S. Sierra‐Aragón2, A. Walker3, J. Timm3, H. Walter4, R. Kaiser2, T. Lengauer1 1
Max‐Planck‐Institut für Informatik, Saarbrücken, Germany Institut für Virologie, Cologne, Germany 3
Universitätsklinikum, Düsseldorf, Germany 4
MVZ Medizinisches Infektiologiezentrum, Berlin, Germany 2
The face of hepatitis C virus (HCV) therapy is changing dramatically with the introduction of directly‐acting antiviral agents (DAAs) that have become widely available since 2011. Despite high response rates of usually more than 90%, treatment failure with DAAs has been associated with the selection of drug resistance mutations which may be present before starting treatment. Additionally, many DAAs show HCV (sub)genotype‐
specific efficacy. Thus, sequence analysis of the viral genome before start of therapy is instrumental for managing DAA‐based treatment strategies. We have created the web‐service geno2pheno[HCV] (g2p) for analyzing HCV sequence data with respect to HCV subgenotype and drug resistance. Extensive reviewing and weighting of HCV drug resistance literature was performed to create a comprehensive list of drug resistance rules for NS3 inhibitors (Boceprevir, Paritaprevir, Simeprevir, and Telaprevir), NS5A inhibitors (Daclatasvir, Ledipasvir, and Ombitasvir), and NS5B inhibitors (Dasabuvir and Sofosbuvir). Upon submission of up to eight sequences, g2p aligns the input sequences, identifies the genomic region(s), predicts the HCV subgenotype(s), and generates a drug resistance report for each DAA. g2p offers convenient and rapid subgenotyping and resistance analysis of HCV sequences. g2p is being continuously updated with state‐of‐the‐art knowledge and is widely used with around 3000 unique queries/month this year. The web‐service is freely accessible under http://www.genafor.org/g2p_hbv/index.php 173 Chronic Viral Infections: HIV and Hepatitis P 145 Immune cell profiles and serum biomarker can predict the outcome of acute HCV infection V. Schlaphoff1, K. Deterding1, S. Hardtke1, J. Hengst1, C. S. Falk2, B. Bremer1, M. Manns1, M. Cornberg1 H. Wedemeyer1 1
2
Medizinische Hochschule Hannover, Klinik für Gastroenterologie, Hepatologie und Endokrinologie, Hannover, Germany Medizinische Hochschule Hannover, IFB‐Tx, Hannover, Germany Background and Aims Acute infection with the hepatitis C virus (aHCV) shows a highly diverse natural course varying between spontaneous clearance of the virus to evolution of persistent infection and chronic hepatitis disease. The aim of this study was to identify easily accessible and reliable parameters during the early time points of aHCV infection which might help in the prediction of the natural course of infection with the prospect of identifying patients who will not achieve spontaneous viral clearance and thus might require antiviral treatment. Methods We performed multianalyte profiling of 50 cytokines and chemokines in the serum of patients using the BioPlex bead array method. Serum samples from 26 patients with acute symptomatic HCV infection recruited within the German Hep‐Net‐acute HCV‐III cohort were analyzed together with ten clinical parameters at the time of diagnosis of infection. In parallel, immune profiles were generated by flow cytometry staining using lymphocytes from the peripheral blood and analyzing the distribution of different immune cell subsets and their activation status. For comparison, we performed the same analyses with samples from healthy controls. To identify profiles characterizing spontaneous sustained viral resolvers we performed principal component analysis (PCA) using Qlucore Omics Explorer Software (Qlucore, Lund, Sweden). Results The well‐described chemokine IP‐10 was analyzed in a first step in order to evaluate its capacity of predicting the natural outcome of aHCV infection. However, serum IP‐10 levels alone did not correlate with the natural outcome of infection. Also, inclusion of IL28b polymorphisms did not correlate with spontaneous clearance. By analyzing all 50 cytokines/chemokines using PCA, several cytokines could be identified that significantly differed between patients achieving spontaneous sustained viral clearance (n=8) from those patients who were still HCV‐RNA positive 12 weeks or more after the first diagnosis of acute HCV infection (n=18). Further, a distinct pattern of cytokine/chemokine expression could be observed for patients achieving viral clearance and those being total non‐responders as well as between patients experiencing an early or late viral relapse. Clinical parameters of liver disease such as serum ALT levels or viral load did not correlate with cytokine levels. The analysis of immune profiles further revealed that the spontaneous clearance of aHCV infection is associated with a profound difference in the frequencies of immune cell populations with an increase of CD4+ T cells and a lower percentage of NK cells as compared to patients who did not spontaneously clear the infection. Conclusion In conclusion, the sustained spontaneous viral clearance of acute HCV infection could be predicted by a specific pattern of serum cytokines/chemokines and a parallel elevated CD4+ T cell/NK cell ratio in the peripheral blood. These parameters might be helpful for the decision whether antiviral treatment of acute HCV infection is required. 174 Chronic Viral Infections: HIV and Hepatitis P 146 Untersuchung des Langzeitverlaufs von Patienten mit einer niedrig‐replikativen chronischen Hepatitis B‐Infektion, die keine antivirale Therapie erhalten: 2 Jahres‐Daten einer deutschen prospektiven Studie (ALBATROS Studie) V. Knop1, E. Herrmann2, J. Vermehren1, J. Petersen3, P. Buggisch3, H. Wedemeyer4, M. Cornberg4, S. Mauss5 M. Sprinzl6, T. Berg7, F. van Bömmel7, H. Klinker8, D. Hüppe9, M. Rausch10, T. Welzel1, A. Vermehren1, S. Susser1 S. Zeuzem1, C. Sarrazin1 1
Klinikum der J.W.Goethe Universität, Medizinische Klinik 1, Frankfurt, Germany J.W.Goethe Universität, Institut für Biostatistik und mathematische Modellierung, Frankfurt, Germany 3
Asklepiosklinik St. Georg, IFI Institut, Hamburg, Germany 4
Medizinische Hochschule Hannover, Hannover, Germany 5
Gastroenterologische Schwerpunktpraxis, Düsseldorf, Germany 6
Universitätsklinik Mainz, I. Medizinische Klinik und Poliklinik, Mainz, Germany 7
Universitätsklinikum Leipzig, Leipzig, Germany 8
Universitätsklinikum Würzburg, Würzburg, Germany 9
Hepatologische Schwerpunktpraxis, Herne, Germany 10
Ärztezentrum am Nollendorfplatz, Berlin, Germany 2
Einleitung Studien aus Asien zeigten, dass die HBsAg‐Konzentration sowie die Ausgangsviruslast prognostisch bedeutsam sind für einen spontanen HBsAg Verlust bei niedrig‐replikativer HBV‐Infektion. Ziel der folgenden Studie war es, die Dynamik von viralen und biochemischen Parametern zu analysieren, welche mit einem HBsAg Verlust bzw einer HBsAg Serokonversion bei inaktiven HBsAg Trägern assoziiert sind. Material und Methoden 672 Patienten mit HBeAg‐negativer HBV‐Infektion wurden prospektiv untersucht. Leitlinienkonform bestand bei keinem Patienten die Indikation zur antiviralen Therapie bei Studieneinschluss. Biochemische und virologische Parameter sowie nicht‐invasive Fibrosemarker wurden zu Baseline sowie 1x/Jahr während des Follow‐up erfasst und zwischen den folgenden Gruppen verglichen: Patienten mit persistierendem positivem HBsAg (Gruppe A), Patienten mit HBsAg Verlust (Gruppe B) und Patienten mit HBsAg Serokonversion (Gruppe C) während der ersten beiden Follow‐up ‐ Jahre. Ergebnisse Verlaufsdaten nach 1, 2 ,3, 4 und 5 Jahren liegen aktuell von 441, 207, 143, 75 und 27 Patienten vor. 12 Patienten haben ihr HBsAg innerhalb von 2 Jahren verloren, und 8 von 12 erzielten eine HBsAg Serokonversion. Patienten ohne HBsAg Verlust (Gruppe A) hatten höhere HBsAg Titer zu Baseline als Patienten mit HBsAg Verlust (Gruppe B) (p<0.001) und HBsAg Serokonversion (Gruppe C) (p<0.0001). In Gruppe B+C betrug der logHBsAg Abfall 2.33 bzw. 3.76 pro Jahr im Vergleich zu ‐0.05 bei den verbliebenen Patienten (n=195) ohne HBsAg Verlust (p<0.0001). Es zeigte sich kein signifikanter Unterschied hinsichtlich des HBsAg Abfalls zwischen Gruppe B+C. Die Höhe der HBV DNA zu Baseline war höher in Gruppe A als in Gruppe B und C (p=0.021 A vs B; p=0.001 A vs C) ohne signifikanten Unterschied der jährlichen Virusdynamik. AST war signifikant höher in Gruppe A vs C (p=0.016). HDL war höher in Gruppe A als in Gruppe B und am niedrigsten in Gruppe C (p=0.046). ALT, GGT, LDL und Gesamtcholesterin zeigten keine signifikanten Unterschiede innerhalb der 3 Patientengruppen. Schlussfolgerung Nach 2 Jahren prospektivem Follow‐up von 672 Patienten mit niedrig‐replikativer chronischer HBV‐Infektion waren HBsAg Verlust und Serokonversionsraten niedrig (6% bzw 4%). Neben der absoluten HBsAg Konzentration zu Baseline war der dynamische HBsAg Abfall ein wesentlicher Parameter zur Vorhersage von HBsAg Verlust und Serokonversion. 175 Chronic Viral Infections: HIV and Hepatitis P 147 CRISPR/Cas9 nickase‐mediated disruption of hepatitis B virus open reading frame S and X J. Schulze zur Wiesch1,2,3, M. Karimova2, N. Beschorner2, J. Chemnitz2, J.‐ H. Bockmann1, W. Dammermann1 D. Indenbirken2, S. Lüth1, A. Grundhoff2,3, F. Buchholz4, J. Hauber2,3 1
Universitätsklinikum Hamburg Eppendorf, Hamburg, Germany Heinrich Pette Institut, Hamburg, Hamburg, Germany 3
DZIF Partner Site, Hamburg, Germany 4
Department of Medical Systems Biology, Dresden, Germany 2
Current antiviral therapies cannot cure hepatitis B virus (HBV) infection; successful HBV eradication would require inactivation of the viral genome, which primarily persists in host cells as episomal covalently closed circular DNA (cccDNA) and, to a lesser extent, as chromosomally integrated sequences. However, novel designer enzymes, such as the CRISPR/Cas9 RNA‐guided nuclease system, provide technologies for developing advanced therapy strategies that could directly attack the HBV genome. For therapeutic application in humans, such designer nucleases should recognize various HBV genotypes and cause minimal off‐target effects. Here, we identified cross‐genotype conserved HBV sequences in the S and X region of the HBV genome that were targeted for specific and effective cleavage by a Cas9 nickase. This approach disrupted not only episomal cccDNA and chromosomally integrated HBV target sites in reporter cell lines, but also HBV replication in chronically and de novo infected hepatoma cell lines. Our data demonstrate the feasibility of using the CRISPR/Cas9 nickase system for novel therapy strategies aiming to cure HBV infection. 176 Chronic Viral Infections: HIV and Hepatitis P 148 Does availability of new DAAs influence treatment uptake in acute hepatitis C in HIV coinfection? C. Boesecke1,2,3, M. Nelson4, P. Ingiliz5, T. Lutz6, S. Scholten7, C. Spinner8, M. Rausch9, T. Reiberger10, S. Mauss11 J. Rockstroh1,2,3 1
Universitätsklinikum Bonn, Medizinische Klinik I, Bonn, Germany German Centre for Infection Research (DZIF), Partner Site Cologne‐Bonn, Bonn, Germany 3
NEAT ID Foundation, London, Great Britain 4
Chelsea and Westminster Hospital, London, Great Britain 5
MiB, Berlin, Germany 6
Infektiologikum, Frankfurt/Main, Germany 7
Praxis Hohenstaufenring, Cologne, Germany 8
Klinikum rechts der Isar, Interdisciplinary HIV Centre (IZAR), Munich, Germany 9
Ärztezentrum Nollendorfplatz, Berlin, Germany 10
Medical University of Vienna, Vienna, Austria) 11
Center for HIV and Hepatogastroenterology, Düsseldorf, Germany 2
Background With the availability of newer DAAs for treatment of chronic hepatitis C infection (HCV) HCV therapy has become considerably less toxic and even more successful than treatment of acute hepatitis C infection (AHC) with pegylated interferon (pegIFN) and ribavirin (RBV). Current DAA‐based regimens are not approved for treatment of AHC and thus, also not reimbursed. Here we evaluate potential changes in the annual rates of treatment initiation in Europe for AHC coinfection in the DAA era. Methodology The PROBE‐C study is an observational European cohort on AHC in HIV coinfection. Between 2007‐2014 483 AHC episodes were documented in 461 HIV‐infected patients from Austria, Denmark, France, Germany, Great Britain and Spain. Fisher's exact, chi‐square and Mann‐Whitney U test were used for statistical analysis. Results All patients were male, median age was 41 years. Main routes of transmission were MSM (97.4%) and IVDU (2.6%). 77.4% of patients were infected with HCV GT1 and 18.9% with GT4. Median baseline HCV‐RNA was 2.019.500IU/mL and median CD4+ T cell count 478 cells/µL. 93% of all patients received cART, 86% had baseline suppressed HIV‐RNA (<200copies/mL). Median ALT was 388 U/l. In 60/483 (12.6%) episodes AHC resolved spontaneously, in 309/483 (64%) treatment with pegIFN/RBV was initiated within 24 weeks of AHC diagnosis. Median time from diagnosis to treatment initiation was 8 weeks. SVR rate was 70%. Overall, as shown in table 1 there was an increase in treatment initiation for AHC from 2007‐2012. However, since 2012 an annual decline from 76% to 45% was observed. There was no association between treatment initiation and HCV transmission risk, HCV GT, HCV RNA levels nor baseline ALT. Conclusions Treatment uptake for AHC has substantially decreased in the last 2 years potentially reflecting patients’ and/or physicians’ wish for a short, well‐tolerated and highly successful DAA‐based therapy which to date is only approved and reimbursed for treatment of chronic HCV infection. However, when treatment during AHC is withhold, more patients remain viremic, which then may foster the epidemic of AHC among HIV‐infected MSM. Therefore, studies evaluating safety and efficacy of IFN‐free DAA regimens for AHC are urgently needed. Figure 1 177 Chronic Viral Infections: HIV and Hepatitis P 149 Redirection of T cells against cells expressing HBsAg ‐ Improving the therapeutic efficiency in preclinical models M. Festag1, K. Wisskirchen1, N. Böttinger2, A. Malo1, F. Bohne1, M. Hudecek3, H. Abken4, U. Protzer1 1
Technische Universität München, Institut für Virologie, Munich, Germany Technische Universität München, Institut für molekulare Immunologie, Munich, Germany 3
Universitätsklinikum Würzburg, Würzburg, Germany 4
Uniklinik Köln, Cologne, Germany 2
Chronic Hepatitis B virus (HBV) infection and HBV‐associated hepatocellular carcinoma (HCC) are a major health concern with currently about 250 million people chronically infected and 0.7 million deaths per year. HBV cannot be eliminated by available antivirals and treatment options for HCC are very limited. Thus, adoptive T cell therapy seems a promising approach not only for chronic hepatitis B but also for HBV‐associated HCC. Previously we generated a chimeric antigen receptor (S‐CAR) recognizing the small HBV envelop proteins S on the surface of infected cells, to graft T cells and target HBV‐infected hepatocytes (Bohne et al., 2008). Using HBV‐transgenic mice, which replicate HBV in hepatocytes, we demonstrated in vivo applicability of this approach. Transferred CD8+ S‐CAR grafted T cells efficiently relocated to the liver causing only transient liver damage with no other obvious side effects and very effectively controlled HBV replication (Krebs et al., 2013). After initial expansion, however, we observed a rapid contraction of the pool of adoptively transferred T cells, and remaining S‐CAR grafted cells showed diminished effector function over time. Therefore, a central goal is to ensure long‐term effector function of S‐CAR‐grafted T cells in vivo. This study aimed at optimizing the S‐CAR construct in a systematic approach. For this, modified S‐CAR constructs were generated harbouring different extracellular spacer domains (IgG1 or IgG4 derived with different lengths or deficient in Fc‐receptor binding) and containing signalling domains from OX40 (CD134) or 4‐1BB (CD137) in addition to that of CD3ζ and CD28. The S‐CARs were stably expressed after retroviral transduction of human T cells. In vitro T cells grafted with most modified S‐CARs did not show a significant difference in their activation potential determined by cytokine secretion upon antigen stimulation. Only the S‐
CARs with a shorter spacer domain displayed a decreased activation potential. Unexpectedly, we observed a decreased killing capacity if a 4‐1BB signalling domain was added. Additional 4‐1BB but even more pronounced additional OX40 signalling induced proliferation of CD8+ T cells, but did not increase activation and cytotoxic potential of S‐CAR grafted T cells. Additional studies will determine which spacer and signalling domain confers longevity to T cells in vivo and is beneficial for the therapeutic outcome. Figure 1 178 Chronic Viral Infections: HIV and Hepatitis P 150 Adoptive T cell therapy with HBV‐specific S‐CAR T cells: Translation to clinical application A. Malo1, K. Wisskirchen1, N. Böttinger2, P. Paszkiewicz3, M. Festag1, U. Protzer1 1
Technische Universität / Helmholtz Zentrum Munich, Institute of Virology, Munich, Germany Technische Universität, Institute of Molecular Immunology, Munich, Germany 3
Technische Universität, Institute of Medical Microbiology, Immunolgy and Hygiene, Munich, Germany 2
Chronic HBV infection is accompanied by a weak T cell response. Current antiviral therapy does not eliminate the virus and cccDNA positive cells, respectively. A possible immunotherapeutic approach is the adoptive transfer of receptor‐modified T cells. We designed a chimeric antigen receptor (CAR) with a single chain antibody fragment recognizing HBV S‐Protein on infected hepatocytes. Primary murine T cells can be grafted with and are activated by a CAR recognizing HBV S Protein on the surface of HBV infected cells. After adoptive transfer, these cells effectively control HBV replication in the liver of HBV transgenic mice. This study is aimed to develop GMP‐compliant protocols for the use of the adoptive T cell therapy in humans. In order to be able to track transduced T cells independently from CAR expression, and to deplete transferred cells on demand, e.g. to prevent a cytokine release syndrome, tEGFR (truncated epidermal growth factor receptor) was added to the transgene cassette. S‐CAR and tEGFR were expressed in parallel in transduced cells, but only the S‐CAR disappeared from the cell surface after antigen contact. When transferred into HBV transgenic mice T cells with high expression of S‐CAR and tEGFR could be depleted using the clinically approved antibody Cetuximab and thereby prevent liver damage. Another step in translation of the S‐CAR approach to clinical application ‐ besides the preclinical evaluation ‐ is the adaptation of retroviral transduction to a large scale, GMP‐compliant protocol. This entails defining optimal growth conditions in terms of media and cytokines, performing all steps in a closed system e.g. bags, and establishing transduction with a self‐inactivating retrovirus to enhance safety. 179 Chronic Viral Infections: HIV and Hepatitis P 151 Impact of genetic polymorphisms in the SCARB1 gene on hepatitis C virus S. Westhaus1,2, M. Deest1,2, F. Stanke3, M. Manns2,4, T. Berg5, C. Sarrazin6, S. Ciesek2,4, T. von Hahn1,2 1
Medizinische Hochschule Hannover, Hannover, Germany Deutsches Zentrum für Infektionsforschung, TTU Hepatitis, Hannover‐Braunschweig, Germany 3
Medizinische Hochschule Hannover, Päd. Pneumologie, Allergologie und Neonatologie, Hannover, Germany 4
Medizinische Hochschule Hannover, Gastroenterologie, Hepatologie und Endokrinologie, Hannover, Germany 5
Universitätsklinikum Leipzig, Hepatologie, Leipzig, Germany 6
Universitätsklinikum Frankfurt, Med. Klinik I, Frankfurt am Main, Germany 2
Background Scavenger receptor type B class I (SR‐BI) being physiologically the main receptor for high density lipoproteins (HDL) on hepatocytes and one essential receptor for hepatitis C virus (HCV) entry is encodes on the SCARB1 gene. A number of single‐nucleotide polymorphisms (SNPs) associated with clinical phenotypes most notably affecting serum lipid levels have been described for SCARB1. Nonetheless, their impact on the HCV replication cycle is unknown. We aimed to investigate SCARB1 SNPs with a high likelihood to affect HCV biology. Methods All known SNPs that result in an amino acid exchange in the SR‐BI protein (5 SNPs) were subjected to HCVcc‐
based in vitro assays testing their ability to function as HCV receptors in Huh‐7.5cells transduced with an SR‐BI specific shRNA for SR‐BI knockdown (7.5/SR‐BIkd) or with CRISPR‐Cas9 for SR‐BI knockout (7.5/TS4‐B2). Furthermore binding of soluble E2 and complete viral particles were analyzed by FACS or quantitative RT‐PCR. Cell surface expression of SR‐BI variants in CHO‐745 cells was analyzed by FACS. Another four non‐coding SNPs that have an allele minor frequency of >10% and had been reported to be associated with a phenotype in humans were investigated in genetic association studies performing single marker and haplotype analyses in a cohort of (n=262) chronically HCV infected patients of the INDIV‐2 study. Results In two out of five coding SNPs we observed lower infectivity compared to wildtype SR‐BI in 7.5/SR‐BIkd and 7.5/TS4‐B2 cells expressing the non‐synonymous SNPs. Comparable infectivity to wildtype SR‐BI was found in cells expressing the other three variants. Soluble E2 (sE2) binding was not detectable in cells expressing mouse SR‐BI and SR‐BI S112F or T175A. However in cells expressing the SR‐BI SNPs G2S, V135I and P297S sE2 binding was detectable. Evaluation of HCVcc binding and the effect of the SNPs on HCV replication is ongoing. Cell surface expression of SR‐BI G2S, V135I or P297S was comparable to human SR‐BI wildtype while surface expression of SR‐BI S112F and T175A was strongly reduced. In the genetic association part of the study we found the G allele of the rs10846744 variant to be associated with higher response rates to dual therapy with interferon and ribavirin in patients chronically infected with genotype 1 of HCV. Furthermore we found an association of the A allele of the rs3782287 variant with higher HCV RNA levels in chronically HCV infected individuals. These results have been confirmed by haplotype analysis and we were able to identify haplotype blocks which could predict treatment outcome and viral load. At the moment we are evaluating expression levels of SR‐BI in human liver tissue from individuals with the respective genotypes. Conclusion Host genetic variation in SCARB1 affect HCV replication in several ways: non‐coding variants seem to modulate the clinical course of HCV infection by affecting the viral load and treatment response. The rare coding variants S112F or T175A showed a decrease in HCV cell entry as result of lower cell surface expression. 180 Chronic Viral Infections: HIV and Hepatitis P 152 Seroprevalence of viral hepatitis E in a population of pregnant women in Brazil S. Hardtke1,2, D. Muzillo3, H. Wedemeyer1,4 1
Medizinische Hochschule Hannover, Gastroenterologie, Hepatologie und Endokrinologie, Hannover, Germany German Centre for Infection Research (DZIF), partner site HepNet Study‐House, Hannover, Germany 3
Universidad Ferderal de Parana, Departamento de Clínica Médica Disciplina de Gastroenterologia Médica, Curitiba, Brazil 4
German Centre for Infection Research (DZIF), partner site Hannover‐Braunschweig, Hannover, Germany 2
Hepatitis E virus genotype 1 and 2 infections are a major cause of death world‐wide and are endemic mainly inSouthern Asiaand Sub‐Saharan Africa. In recent years, the importance of autochthonous hepatitis E genotype 3 infection has been recognized in industrial countries with severe chronic infections in immunocompromized individuals and serious HEV‐associated autoimmune disorders. Genotype 1 hepatitis E may take particular severe courses in pregnancy with high maternal and fetal mortality rates. The anti‐HEV prevalence and HEV genotype distributions inSouth Americaare not well defined. Small studies fromBrazilreported HEV seroprevalence rates of up to 10% in blood donors, slightly higher numbers in individuals from an agricultural settlement in theAmazonBasinand 15% in renal transplant recipients. There are no reports on disease severity in special risks groups. Objectives To investigate the HEV seroprevalence and frequency of acute hepatitis E in pregnant women inSouthern Brazil Methods Sera from 295 pregnant women and 221 female blood donors were collected from 2002 to 2003 in the Hospital de Clínicas, Universidade Federal do Paraná. In this DZIF‐sponsored HepNet Study‐House project 421 samples were shipped to Hannover in 2014 and tested for anti‐HEV IgG antibodies with the WANTAI‐ ELISA Assay. Samples tested positive for anti HEV IgG were tested for anti‐HEV IgM and HEV RNA. All samples were also tested for HBsAg, anti‐HBc and anti‐HCV. Results A total of 35 of 214 pregnant women and 50 of 207 samples in the blood donor group tested positive for anti‐
HEV IgG corresponding to seroprevalences of 16 % and 24% (Fisher´s exact test p=0.13). Importantly, HEV‐RNA was negative in all samples. The mean age of anti‐HEV‐positive and anti‐HEV‐negative women did not differ in either group and was also not different between pregnant women and blood donors (mean age of 28.3 years vs. 29.0 years, respectively), however, the proportion of anti‐HEV‐positive results was higher in blood donors older than 35 years (30.6%) than in younger (<25 years) donors (11.6%) . White pregnant women tested less often anti‐HEV positive than women with other races (12.9% vs. 20.4%). Only 1 sample from pregnant women and 2 of the donor group tested positive for HBsAg while 25 pregnant women and 6 of blood donors were anti‐HBc positive. None of the women tested positive for anti HCV. Conclusion This first study investigating the seroprevalence of anti‐HEV in in pregnant women inBrazilshowed higher antibody frequency than previous reports fromBraziland similar frequencies as in centralEurope. Importantly no sample tested positive for HEV RNA indicating that acute HEV infection is not a major threat for pregnant women in Brazil. Still, a significant proportion of individuals become HEV infected during life‐time also in South America. More studies are needed to determine the health burden associated with these infections. 181 Chronic Viral Infections: HIV and Hepatitis P 153 Hepatitis C Virus Screening Project of Patients on current anti‐HCV Therapy S. Sierra1, E. Knops1, P. Kalaghatgi2, M. Neumann‐Fraune1, T. Lengauer2, V. Keitel3, T. Goeser4, S. Esser5 T. von Hahn6, I. Peuser7, N. Qurishi8, S. Scholten9, M. Däumer10, R. Kaiser1 1
Institut für Virologie, Cologne, Germany Max Planck Institute for Informatics, Saarbrücken, Germany 3
University Hospital Düsseldorf, Düsseldorf, Germany 4
University Hospital Cologne, Cologne, Germany 5
University Hospital Essen, Essen, Germany 6
Medizinische Hochschule Hannover, Hannover, Germany 7
7Märkische Radioonkologische Versorgungszentren GmbH, Lüdenscheid, Germany 8
Private Practice Gotenring, Cologne, Germany 9
Private Practice Hohenstaufenring, Cologne, Germany 10
Institute of Immunology and Genetics, Kaiserslautern, Germany 2
Background Clinical outcome of HCV therapy of direct acting antivirals (DAAs) depends on host and viral factors. This observational, retrospective and non‐interventional study collects data from DAA‐resistance‐associated‐
mutations (RAMs) within the viral NS3/protease, NS5A and NS5B genes, their viral quasispecies distribution and host factors, to predict clinical outcome using the geno2pheno[HCV] tool. Material and methods Viral NS3/protease, NS5A and NS5B sequences from plasma samples were obtained. Subtyping and resistance against Boceprevir (BOC), Telaprevir (TVR), Simeprevir (SMV), Daclatasvir (DCV), Sofosbuvir (SOF) and Ledipasvir (LDV) was determined by sequencing of the corresponding viral gene and subsequent interpretation with geno2pheno[HCV] (http://hcv.bioinf.mpi‐inf.mpg.de/). Baseline samples from patients under following therapies were analysed: IFN/RBV (49); IFN/RBV/TVR (74); IFN/RBV/BOC (31); IFN/RBV/SOF (30); RBV/SOF (34); RBV/SMV (2); RBV/SOF/DCV (5); SOF/SMV (9); SOF/DCV (8); SOF/LDV (31). Results 647 HCV‐infected patients have been enrolled until end of January 2015. Enrollment increased sharply up to 2014, when the new DAAs were licensed. We produced baseline sequences of NS5B and/or NS3/protease regions from 537/647 patients. Therapy information and viral load values from 411/537 patients was obtained. Geno/subgenotyping (GT) was successful for 353/537 baseline‐samples: 104 GT1a (clade I), 71 GT1a (clade II), 143 GT1b, 15 GT2, 65 GT3a, 16 GT4. 251 NS3/protease baseline samples could be sequenced. We analyzed the presence of mutations in the amino acid positions 36, 41, 54, 55, 80, 87, 117, 122, 132, 155, 156, 168, 170 and 174, which have been described to be involved in PI‐resistance. In 165/251 (65.7%) samples RAMs were detected; the most common ones were 132V 55/251 (21.9%); 80K 33/251 (13.1%); 122G 10/251 (4.0%). 353 NS5B baseline samples were successfully sequenced. The amino acid positions 15, 96, 179, 289, 282, 293, 316, 414, 415, 423, 434, and 479, described to be involved in SOF‐resistance, were checked. In 113/353 (32.0%) samples RAMs were detected (most frequent ones: 415Y+479P 35/353 (9.9%); 293L+479P 21/353 (5.9%); 293L+415Y+479P 9/353 (2.5%); 293L+415Y+434L+479P 9/353 (2.5%). Furthermore, we could generate 26 NS5A baseline samples. We scrutinized the amino acid positions 23; 28; 30; 31; 32; 58; 92; 93, related to DCV/LDV‐resistance. In 9/26 samples (34.6%) RAMs were detected (58P 2/26 (7.7%); 30R+58P 4/26 (15.4%); 28L+30R+58P 3/26 (11.5%). Conclusion Analysis of NS5B, NS5A and NS3/PR with geno2pheno[HCV] interpretation allows subtyping, clade classification, and prediction of DAAs susceptibility. PI resistance‐mutations exist at baseline in higher extent as reported in previous studies. Incorporating additional viral and clinical data will continuously improve geno2pheno[HCV] for better predictions. 182 Chronic Viral Infections: HIV and Hepatitis P 154 shRNA sense strand neutralization reduces off‐targeting, ameliorates toxicity and enhances efficacy of RNAi‐based gene therapy for chronic hepatitis B T. Michler1, S. Grosse2, S. Mockenhaupt2, N. Röder1, F. Stückler3, B. Knapp3, M. Heikenwälder1, D. Grimm2 U. Protzer1 1
Technische Universität München, Virologie, Munich, Germany Universitätsklinikum Heidelberg, Virologie, Heidelberg, Germany 3
Helmholtz Zentrum München, Munich, Germany 2
Current therapy for chronic hepatitis B patients is unsatisfying, as nucleoside‐analogs don’t eliminate the virus and often need to be given life‐long. Also, while preventing the generation of infectious HBV virions, nucleoside‐analogs fail to suppress HBV surface and e‐antigen, which enable viral persistence by modulating the host immune system and contribute to HBV‐associated hepatocellular carcinoma. These problems may be solved using RNAi as anti‐HBV therapeutic since (i) long‐term suppression can be achieved by a single dose of an shRNA‐encoding vector, and (ii) all four HBV transcripts share a common 3´end, allowing concurrent inhibition of HBV pre‐genomic RNA and all viral proteins with a single RNAi molecule, and potentially restoring antiviral immunity and preventing carcinogenesis. Still, clinical translation remains hampered by safety concerns as shRNA over‐expression in the mouse liver causes elevated liver transaminases, jaundice and weight loss. One explanation is that ectopic RNAi triggers overload the endogenous miRNA pathway, perturbing miRNA biogenesis and/or activity, and causing cytotoxicity. Evidence for this saturation model is that (i) shRNAs circumvent Drosha processing, a gatekeeper for miRNA biogenesis, (ii) embedding shRNAs in a recombinant miRNA context yields lower and safer levels of mature RNAi triggers, and (iii) overexpression of Argonaute‐2 (Ago2) ameliorates toxicity and enhances RNAi efficacy. Moreover, RNAi triggers can perturb cell physiology through unwanted inhibition of off‐target genes with partial complementarities to one of the two strands of the RNAi molecule. To alleviate such unintended gene silencing, we have developed a novel bi‐cistronic AAV vector that expresses, in addition to the shRNA, a second RNA hairpin called “tough decoy” or TuD. In cell culture, the TuD effectively sequestered and inactivated shRNA sense strands, and thereby improved RNAi specificity. These remarkable features translated well into an HBV‐transgenic mouse model, where sense strand off‐target activity was likewise prevented, as validated by transcriptome analysis of liver RNA, coinciding with ameliorated toxicity. Enhanced in vivo safety was also noted for two alternative AAV vectors, either co‐
expressing the shRNA and Ago2, or embedding the shRNA in a miR‐122 backbone. Notably, the new shRNA/TuD vector outperformed the other expression strategies regarding efficacy with stable HBV reduction of up to 98% over 3 months. We speculate that its enhanced antiviral efficacy results from increased loading of the desired antisense strand into RISC in absence of the sense strand, possibly also further amending toxicity by attenuating RISC saturation. In conclusion, we show how unraveling the mechanisms of RNAi‐toxicity and the rational design of new expression vectors enables the design of safe and efficient RNAi‐gene therapy for chronic hepatitis B. 183 Chronic Viral Infections: HIV and Hepatitis P 155 Kinetic of myeloid‐derived suppressor cells in chronic HIV‐1 infection E. Grützner1, R. Stirner1, L. Arenz1, A. Athanasoulia2, K. Schrödl3, J. Bogner1, R. Draenert1 1
Klinikum der Universität München, Sektion Infektiologie, Medizinische Klinik und Poliklinik IV, Munich, Germany Klinikum der Universität München, Medizinische Klinik und Poliklinik IV, Munich, Germany 3
Klinikum der Universität München, Medizinische Klinik und Poliklinik V, Munich, Germany 2
Background Myeloid derived suppressor cells (MDSC) have been described as a group of immature myeloid cells which exert immunosuppressive action by inhibiting function of T lymphocytes. Initially discovered in a variety of solid tumors, MDSC were recently also found in infectious diseases e.g. in tuberculosis and HIV. There are two main subgroups of MDSC: granulocytic MDSC (gMDSC) and monocytic MDSC (mMDSC). So far, there is limited knowledge concerning the time point when analyses are best made after blood draw. To further elucidate the significance of MDSC in HIV infection in future experiments, we aim at standardizing the analytical process. As one critical aspect we defined the time frame between blood draw and cell analysis. Methods In this study, we isolated peripheral blood mononuclear cells (PBMC) from fresh blood directly after blood draw. Part of the cells were frozen. We then analyzed the frequencies of both gMDSC and mMDSC 2, 4 and 6 hours after blood draw and after an overnight rest by FACS analysis. In addition we measured MDSC after thawing the frozen PBMC. Results For granulocytic MDSC (gMDSC), our results showed no significant difference in gMDSC frequencies using fresh PBMC over time (i.e. at the time points 2h, 4h, 6h, and overnight). However, gMDSC levels were drastically reduced after freezing the PBMC (p<0.0001). In contrast, frequencies of monocytic MDSC (mMDSC) varied during the course of time. There was no difference between time point 2h and 4h. But there was a significant reduction at time point 6h and overnight (p=0.0005 and p=0.005 respectively). Freezing of PBMC reduced mMDSC not as dramatically as gMDSC, but the decrease still reached statistical significance (p=0.039). For both MDSC subgroups, FACS analysis became more difficult over time due to less sharp divisions between populations. Conclusion According to our data MDSC need to be studied on fresh PBMC. gMDSC can be studied with delay, mMDSC however should be studied no later than 4h after blood draw. These results are crucial as even many published studies make use of frozen PBMC when analyzing MDSC. 184 Chronic Viral Infections: HIV and Hepatitis P 156 Designing viral vectors for blocking HIV‐1 in brain cells. C. Kammel1, E. Kienle2, F. Schmidt2, M. Schneider1, D. Grimm2, R. Brack‐Werner1 1
2
Helmholtz Center Munich, Virology, Munich, Germany Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany The main obstacle in curing HIV‐1 infection is the persistence of long‐lived cells with functional and often latent HIV‐1 genomes. These virus reservoirs reside in different organs and are formed by various cell types like resting CD4+ T‐cells, macrophages and astrocytes. Current therapeutic strategies for elimination of resting CD4+ T‐cell reservoirs aim to deliberately kill reservoir cells, which are expected to be naturally replenished by uninfected cells. However, this strategy is not feasible for brain cells, because of the very low cell turn‐over in this organ. Instead we propose to design viral vectors that prevent persistence and/or expression of HIV‐1 genomes in brain cells. A library consisting of wildtype and artificially modified AAVs (Adeno‐associated virus) was screened to identify AAV serotypes that are able to transduce the neural stem cell line HNSC.100, which can be differentiated into astrocytes. The identified serotype AAV9P1 in combination with a truncated GFAP promoter (GFAP = astrocytic marker) was tested for its ability to transduce HNSC.100 cells and other cell types including T‐cells and liver cells. We show that the AAV9P1_GFAP‐YFP vector transduces neural stem cells and astrocytes. No transgene expression was detected in all other tested cell lines. The AAV9P1_GFAP‐YFP vector was able to introduce a long‐term transgene expression in differentiated HNSC.100 cells, which was detectable on DNA‐ and protein level even 70 days post transduction. In order to target and inactivate the HIV‐1 genome in latently infected cells, the genome editing system CRIPSR/Cas9 was used. A screening system was established to identify potent gRNA sequences for disruption of the HIV‐1 infection. Identified gRNAs were used for AAV production. We show that transduction with CRISPR/Cas9 AAVs significantly reduced reactivation of HIV‐1 expression in latently infected, differentiated HNSC.100 populations, which were exposed to TNF‐α. Taken together we could show that the AAV9P1 vector system is a potent tool for targeting HIV‐1 infected cells in the brain. 185 Chronic Viral Infections: HIV and Hepatitis P 157 The Cyclosporine A (CsA) Washout assay allows to assses the capsid stability and study uncoating of HIV‐1 P. Schommers1,2, M. Altfeld2 1
2
University of Cologne, Infektiologie, Cologne, Germany Heinrich‐Pette‐Insitut, Department of Virus Immunology, Hamburg, Germany Background At early stages during the HIV‐1 replication and after the viral particle fused with the targeted cell, the conial capsid dissociates in a process known as uncoating. The exact timing and which factors contribute to uncoating are still poorly understood. Earlier work has shown that the stability of the capsid and the time point at which it uncoats predicts the infectivity of the viral particle. However it is not completely known why and how the capsid affects the viral fitness. Therefore we adopted the cyclosporine A washout assay that was first published by Hulme et al. in our lab to study the impact of mutations in Gag on the stability of the capsid stability and its uncoating. Method The CsA washout assay indirectly allows us to draw conclusions about the capsid stability. It is a cell‐based assay that uses the HIV‐1 restriction factor tripartite motif‐cyclophilin A (TRIM‐CypA) in order to detect and inhibit the infections of viral complexes that are coated by p24 capsid. TRIM‐CypA is known to inhibit the viral replication by binding the conical capsid and so preventing the uncoating. The drug CsA is able to block the interaction of TRIM‐CypA with the capsid, which allows the virus to uncoat again and infect the cell. Owl monkey kidney (OMK) cells, that endogenously express TRIM‐CypA, get infected by VSV‐g pseudotyped HIV‐
GFP reporter virus in the presence of CsA. At various time‐points after the infection, CsA is washed out and every viral complex that is uncoated is able to reach the nucleus, integrate and initiate replication. Cells in which HIV‐1 replicates express GFP can then be quantified by FACS. The percentage of the GFP+ cells allows to calculate the percentage of uncoated virions in the assay at the time point of the CsA washout. This allows conclusions about the stability of the capsid as an unstable capsid will uncoat faster and the viral complex will infect more cells at a certain time point of the CsA washout. In Fig.1 an example of the CsA washout assay is shown in which we assessed the process of uncoating with a wild type NL4‐3env‐GFP reporter virus that has a frameshift in env and expresses GFP in place of nef. It shows the increasing percentage of GFP+ OMK cells over time in the CsA‐washout group. Later time points have a higher infection rate as the virus had more time to uncoat and got integrated before the CsA got washed out. To serve as a control the same procedure was done with ethanol (EtOH) instead of CsA. Almost no infection was seen in the control group as the Trim‐CypA of the OMK cells inhibited uncoating and thereby integration. Aim This assay allows us to determine the mechanisms by which viral cytotoxic T‐cell escape mutations in p24 Gag can result in enhanced sensing of HIV‐1 by the innate immune system, and modulate the sensitivity of the virus to host restriction. We will test the hypothesis that viral escape mutations within HIV‐1 capsid resulting from CTL‐mediated immune pressure destabilize capsid and lead to more rapid sensing of viral oligonucleotides by innate immune receptors. Figure 1 186 Chronic Viral Infections: HIV and Hepatitis P 158 Latent HIV‐1 Reactivation and Autophagy Inhibition Synergize to Host Cell Death M. Stankov1, C. Suhr2, H. Lin1, D. Panayotova‐Dimitrova3, C. Goffinet 2, G. Behrens1 1
Hannover Medical School, Department for Clinical Immunology and Rheumatology, Hannover, Germany TWINCORE, Experimental virology, Hannover, Germany 3
Mannheim Clinic of University of Heidelberg, Department of Dermatology, Venerology and Allergology, Mannheim Germany 2
Introduction Current strategies targeting latent HIV‐1 reservoir eradication ‘the shock and kill approach’ rely mostly on pharmacological reactivation followed by elimination of cells that reactivated viral latency by either the virus or/and patient's immune system. So far, most of these approaches demonstrated only incomplete reactivation and reservoir reduction. Objectives We aimed to develop a pharmacological strategy targeting cellular pro‐survival mechanisms in order to sensitize and eliminate host cells with even incomplete levels of HIV‐1 reactivation. Materials & Methods We used in vitro models of HIV latency (J‐lat full length clones, ACH2 and U1) and the latency‐reversing agent panobinostat (Pan). We targeted late stages of the pro‐survival pathway of autophagy using lysosomotropic agents such as chloroquine (CQ). Autophagy and lysosomal pathways were monitored using flow cytometry, fluorescent microscopy and western blotting. Latent HIV‐1 reactivation was confirmed through HIV‐1‐GFP expression, intracellular p24 abundance, and secretion. Results Our results confirmed CQ mediated suppression of autophagic flux. Pan mediated reactivation led to specific cell death, which closely correlated to the levels of reactivation as measured by HIV‐GFP MFI (R2=0.8821; p<0.02) and changes in HIV‐GFPhigh population (R2=0.8724; p=0.02). Importantly, single round reactivation (0.1µM Pano for 18h) failed to eliminate the HIV‐1 reactivating cells even after 62h in culture. However, CQ treatment introduced upon reactivation led to the complete eradication of all HIV‐1 reactivating cells (including those with low or incomplete levels of reactivation) in only 34h with almost no toxicity to HIV‐1 non‐producing cells. Conclusion We conclude that deprivation of the pro‐survival pathway of autophagy through inhibition of its late stages of lysosomal function efficiently facilitates host cell death upon incomplete HIV‐1 reactivation. These in vitro results suggest that combined pharmacological intervention may assist to eradicate latently HIV‐1 infected cells. 187 Chronic Viral Infections: HIV and Hepatitis P 159 Terminal differentiation of CD56dimCD16+ NK cells is associated with increase in NK cell frequencies after ART in HIV‐1 infection F. Ahmad1, D. M. Tufa1, M. Neha1, R. Jacobs1, R. E. Schmidt1 1
Medizinische Hochschule Hannover, Clinical Immunology, Hannover, Germany Background HIV‐1 infection is known to have a detrimental impact on natural killer (NK) cell phenotype and functions. In this study, we investigated the effect of antiretroviral therapy (ART) on distributions, activation and terminal differentiation of the NK cell subsets in a longitudinal study. Methods We analyzed NK cell subsets by flow cytometry from cryopreserved peripheral blood mononuclear cells (PBMCs) in 35 untreated HIV‐infected individuals, 25 treated HIV‐infected individuals and 15 healthy subjects. 21 HIV‐infected patients were longitudinally followed before and after 1 year of initiation of ART. Results We found that frequencies as well as absolute counts of CD56bright CD16+ NK cells in HIV+ blood donors were similar compared to uninfected controls. CD56dimCD16+ NK cells were decreased while CD56negCD16+ NK cells were expanded in untreated HIV‐infected patients as compared to uninfected controls. Treated patients had higher frequencies of CD56dimCD16+ NK cells and lower frequency of CD56negCD16+ NK cells as compared to untreated patients. The decrease in the frequencies of CD56dimCD16+ NK cells in untreated patients was inversely correlated with CD56negCD16+ NK cell expansion. Frequencies of CD69+/CD56dimCD16+ NK cells and CD69+/CD56negCD16+ NK cells restored after ART. Terminal differentiation of CD56dimCD16+ NK cells is enhanced after ART as measured by CD57 expression. In the longitudinal study, we observed that increase in the frequency of CD57+CD56dimCD16+ NK cells only in those patients, which also have an increase in absolute NK cell counts/frequencies after the treatment. Frequencies of CD57+CD56dimCD16+ NK cells is directly correlated with the frequencies of total NK cells while frequencies of CD56dimCD16+ NK cells were not correlating with NK cell frequencies, suggesting that increase in the frequencies of CD57+CD56dimCD16+ NK cells is reflected by increased frequencies of total NK cells after ART. Conclusion Our data indicate that ART has an effect on immune restoration of NK cells and enhanced in the terminal differentiation of CD56dimCD16+ NK cells is associated with increased frequencies of total NK cells after ART. (DZIF TTU 04.802 and HBRS support this study) 188 Chronic Viral Infections: HIV and Hepatitis P 160 PQBP1 is Required for Innate Immune Response to HIV‐1 Infection in DCs and is a Specific Sensor of Retroviral DNA J. Seifried1, H. Schmitz1, C. Tondera1, S. M. Yoh2, D. Germanaud3, V. Des Portes4, K. Cichutek5,6, S. K. Chanda2 R. Koenig2,1,6 1
Paul‐Ehrlich Institut, Host Pathogen Interactions, Langen, Germany Sanford Burnham Prebys Medical Discovery Institute, Immunity and Pathogenesis Program, La Jolla, USA 3
AP‐HP, Hopital Robert Debre , Service de Neurologie Pediatrique et des Maladies Metaboliques, Paris, France 4
Hospices Civils de Lyon, Universite de Lyon, National Reference Center for Fragile X and Other XLID; Biobank Neurobiotec, Bron, France 5
Paul‐Ehrlich‐Institut, Langen, Germany 6
Deutsches Zentrum für Infektionsforschung (DZIF), Langen, Germany 2
In recent years, the critical role of the innate immune system for the elicitation of an effective immune response to human immunodeficiency virus type 1 (HIV‐1) has become an emerging concept. Understanding the complexity of how innate responses affect the outcome of HIV‐1 infection will help in the development of novel HIV vaccine strategies. Dendritic cells (DCs) play a critical role in the immune response to viral infection through the facilitation of cell‐
intrinsic antiviral activity and the activation of adaptive immunity. HIV‐1 infection of DCs triggers an IRF3‐
dependent innate immune response, which requires the activity of cyclic GAMP synthase (cGAS). We report the identification of a novel immune regulator, polyglutamine binding protein 1 (PQBP1), that directly binds to HIV‐1‐encoded reverse‐transcribed HIV‐1 DNA to initiate the cGAS dependent innate response and thus acts as a critical front‐line sensor for the initiation of an immune response to HIV. We propose a model in which cGAS, by virtue of its association with specific co‐receptors, such as PQBP1, increases its ability to recognize distinct molecular patterns encoded by pathogen‐associated DNA. Renpenning syndrome is caused by mutations in the PQBP1 locus, a severe X‐linked mental retardation syndrome associated with intellectual deficiency, microcephaly, short stature and facial dysmorphy. The disease is very rare, with a total of 52 published cases world‐wide. We investigated DCs of two male patients who harbor a genetic mutation in the PQBP1 locus that results in a C‐terminal truncation of amino acids 153‐
265 in the protein, leading to a loss of reverse‐transcribed HIV‐1 DNA binding. MDDCs derived from the Renpenning patients possess a severely impaired innate immune response to HIV‐1 challenge, underscoring the role of PQBP1 as a proximal innate sensor of HIV‐1 infection: they display an almost undetectable induction of ISG54 in comparison to those of infected healthy donors, despite comparable viral infectivity. However, MDDCs derived from Renpenning patients display a comparable innate immune response in comparison to healthy donors towards Sendai virus, MVA, or herring testis (HT) DNA, showing that the innate immune response to an RNA virus, a DNA virus or HT DNA is not dependent upon PQBP1. As PQBP1 and cGAS seem to form a pattern‐recognition receptor complex specifically responding to retroviral PAMPs, the HIV‐1 encoded PAMP is likely based on a unique and discernable secondary structure only generated during the RT step. Identifying the exact structure of the PAMP would give rise to a promising new target for a vaccine adjuvant that could advance the development of HIV vaccines or therapeutic strategies. 189 Chronic Viral Infections: HIV and Hepatitis P 161 Measles, mumps, rubella and VZV: importance of serologic testing of vaccine preventable diseases in young adults living with HIV in Germany C. Schwarze‐Zander1, R. Draenert2, C. Lehmann3, C. Boesecke1, S. Sammet2, J.‐ C. Wasmuth1, U. Seybold2 D. Gillor3, U. Wieland4, T. Kümmerle3, C. P. Strassburg1, A.‐ M. Eis‐Hübinger5, A. Mankertz6, G. Fätkenheuer3,7 J. Bogner2, J. Rockstroh1, J. J. Vehreschild3,7 1
University Hospital Bonn, Medizinische Klinik I, Bonn, Germany Klinikum der Universität München, Medizinische Klinik und Poliklinik IV, Munich, Germany 3
Uniklinik Köln, Klinik I für Innere Medizin, Cologne, Germany 4
Uniklinik Köln, Institut für Virologie, Cologne, Germany 5
University Hospital Bonn, Institut für Virologie, Bonn, Germany 6
Robert Koch Institute, Division of Viral Infections, Berlin, Germany 7
German Centre for Infection Research, Partner Site Bonn‐Cologne, Cologne, Germany 2
Background Measles, mumps, rubella (MMR) and varicella infection can cause serious diseases and complications in the HIV positive population but can be prevented by vaccination. Due to successful vaccination programs measles, mumps and congenital rubella syndrome have become uncommon in Germany. However, recent outbreaks of measles have occurred from import‐associated cases in Germany. We evaluated MMR and VZV humoral immunity in a German cohort of HIV positive adults and aimed to identify associated factors for lack of MMR and VZV seroprotection. Methods In this prospective, cross‐sectional study the serostatus for MMR and VZV in a cohort of people living with HIV in Germany was analyzed. Sera were tested for MMR and VZV specific immunglobulin G antibodies using commercial immunoassays. Results This study analyzed 2013 HIV positive adults from 3 different University out‐patient clinics in Bonn (n=544), Cologne (n=995) and Munich (n=474). In this population insufficient seroprotection was found in 3% for measles, 26% for mumps, 11% for rubella and 2% for VZV. Regarding MMR 35% of patients were lacking sufficient immunity against at least one entity. In multivariable analysis younger age was strongly associated with insufficient immunity against all 4 entities, MMR (p<0.001, p<0.001 and p=0.01, respectively) and VZV (p<0.001). Conclusion In people living with HIV in Germany there is high need for MMR and VZV vaccination in those born after 1970. Thus, systematic MMR and VZV antibody screening and vaccination should be implemented in the HIV positive population in Germany to prevent serious disease and complications of vaccine‐preventable diseases. 190 Chronic Viral Infections: HIV and Hepatitis P 162 Characterization of CD3+CD56+ Natural Killer‐like T cells in HIV+ patients with acute hepatitis C P. Kokordelis1,2, B. Krämer1,2, C. Boesecke1,2, A. Glässner1,2, C. Schwarze‐Zander1,2, E. Voigt3, P. Ingiliz4, P. Lutz1,2 F. Goeser1,2, U. Spengler1,2, J. Rockstroh1,2, J. Nattermann1,2 1
Universitätsklinikum Bonn, Medizinische Klinik und Poliklinik I, Bonn, Germany Deutsches Zentrum für Infektionsforschung, Bonn, Germany 3
Praxis am Ebertplatz, Cologne, Germany 4
Medical Center for Infectious Diseases (MID), Berlin, Germany 2
Background Co‐infection with HCV is a major cause of morbidity and mortality in HIV(+) patients. CD3+CD56+ natural killer‐
like T cells, a subset of T lymphocytes, have been suggested to modulate the outcome of acute HCV mono‐
infection. However, the role CD3+CD56+ natural killer‐like T cells in HIV+ patients with acute hepatitis C remained unclear. Methodology 36 HIV (+) patients with acute hepatitis C (self‐limiting, n=13; chronic course, n=23) were studied. As controls eight HIV mono‐infected patients, 12 HIV(+) patients with chronic hepatitis C, eight HIV(‐) patients with chronic HCV infection as well as 12 healthy individuals were analyzed. Peripheral NKT cells (CD3+CD56+) were analyzed by flow‐cytometry. IFN‐γ secretion and anti‐HCV activity of NKT cells were analyzed using the HuH7 HCV replicon system. Results Frequency of CD3+CD56+ NK‐like T cells did not differ significantly between the groups.However, we observed a significant higher expression of the maturation markers CD27, CD127, and CD161 on CD3+CD56+ NK‐like T cells in healthy controls than in HIV mono‐infected patients and HIV patients with an acute HCV infection, respectively. Interestingly, CD3+CD56+ NK‐like T cells from HIV patients with acute hepatitis C displayed a higher CD69 expression, indicating a more activated status as compared to healthy controls. Of note, a high CD69 expression was associated with spontaneous clearance of acute hepatitis C, suggesting a role for CD3+CD56+ NK‐like T cells in anti‐HCV immune responses. Accordingly, we found IL12‐activated CD3+CD56+ NK‐like T cells to effectively block HCV replication in an IFN‐g dependent fashion. However, CD3+CD56+ NK‐like T cells from HIV patients with acute HCV infection displayed a significantly impaired IFN‐g production compared to both HIV mono‐infected as well as healthy individuals. Conclusion Our results indicate that CD3+CD56+ NK‐like T cells have the potential to block HCV replication but are functionally impaired in HIV+ patients with acute hepatitis C. 191 Chronic Viral Infections: HIV and Hepatitis P 163 Cell Surface Downregulation of NK cell Ligands by Patient‐derived HIV‐1 Vpu and Nef Alleles J. Galaski1, F. Ahmad2, N. Tibroni1, R. E. Schmidt2, O. Fackler1 1
2
University Hospital Heidelberg, Integrative Virology, Heidelberg, Germany Hannover Medical School, Department of Clinical Immunology and Rheumatology, Hannover, Germany Objective HIV‐1 Vpu and Nef proteins downregulate cell surface levels of NK cell ligands but functional consequences of individual downregulation events are unclear. We tested how well conserved NK cell ligand downregulation is among Vpu and Nef variants isolated from chronic HIV patients and whether their downregulation activity is linked to disease progression. Methods Proviral vpu and nef sequences were amplified from 27 chronic HIV patients, subcloned, and tested for their ability to downregulate cell surface receptors. Downregulation activities were correlated to clinical patient parameters. Results Cell surface downregulation of CD4, CD317/tetherin and MHC‐I that exert biological functions other than NK cell activation were well conserved among patient derived Vpu and Nef variants. Among NK cell ligands, NTB‐A, PVR and ULBP were identified as main targets for downregulation with specific sensitivity to Vpu (NTB‐A, PVR, ULBP) or Nef (PVR). Downregulation of cell surface ULBP was identified as a novel and highly conserved activity of HIV‐1 Vpu but not Nef. NK cell ligand downregulation did not correlate with viral load, CD4 or NK cells counts, or NK cell activation in the respective patient. Conclusions The conservation of downregulation of major NK cell ligands by either HIV‐1 Vpu or Nef suggests an important pathophysiological role of this activity that may impact on the acute phase of HIV infection. 192 Chronic Viral Infections: HIV and Hepatitis P 164 High frequencies of CD39‐positive Natural Killer cells during HIV infection correlate with disease progression parameters and regulate effector functions by degrading extracellular ATP P. Dierks1, J. Eberhard1, J. Schulze Zur Wiesch1 1
University Medical Center Hamburg Eppendorf, I. Medical Department, Gastroenterology and Infectius Diseases, Hamburg, Germany Functional NK cell responses have been shown to be relevant during the coordinated immune response against chronic viral infections like HIV. NK cell activation is not regulated by a dominant receptor as in B or T cells, but is the result of a balance of various signals recognized by C type lectin‐like and Killer‐cell immunoglobulin‐like receptors. The ectonucleotidases CD39 and CD73 are known for its ability to degrade pro‐inflammatory extracellular ATP to anti‐inflammatory Adenosine. This ability is involved in immune cell regulation in an autocrine and paracrine manner. Recently, dysregulations of this purinergic pathway have been shown to affect Treg and CD8 T cell responses during HIV. As ATP and Adenosine are known to regulate NK cell responses, the aim of this study is to reveal the expression pattern of CD39 on NK cells and its functional consequences during HIV infection. Therefore, we analyzed PBMC samples derived from a cohort of HIV‐infected treatment‐naïve (n=30), treated (n=20), long term non‐
progressor (n=6), HIV/HCV‐coinfected (n=6) patients and compared them to healthy controls (n=20) via multicolor FACS. We found significantly increased amounts of CD39‐positive NK cells in HIV compared to healthy controls, with the highest levels in treatment‐naïve individuals (Abb.1). The expression level of CD39 on NK cells correlated with disease progression parameters like CD4 count (inverse correlation, R²=0.62, p=0.0001; Abb.2) and CD8 T cell activation (R²=0.29, p=0.01) during untreated HIV. The levels of CD73 remained unaltered. Furthermore, we detected a differentiation‐associated skewing of CD39 expression on NK cells with high levels on immature NKG2A+ CD56bright NK cells compared to mature KIR+CD57+ CD56dim NK cells in all groups. We confirmed the assumption that CD39‐positive NK cells are able to degrade ATP in contrast to CD39‐negative NK cells by a Luciferase‐based ATP detection assay. Additionally, we observed that levels of functional CD39 on NK cells are highly inducible by activation with cytokines like IL2 and/or IL12. Functionally, we observed a higher proliferative capacity and higher production of IFNgamma in the CD39‐positive NK cell compartment after stimulation. Furthermore, we inhibited proliferative NK cell responses and IFNgamma production by adding different amounts of ATP. Consequently, we found that CD39‐positive NK cells were significantly less sensitive to ATP‐mediated inhibition of effector functions. We provide first evidence that NK cells express high amounts of CD39 during HIV infection. This expression seems to be differentiation‐associated and influences NK cell functions under presence of pro‐inflammatory ATP, which may have consequences for NK responses and course of HIV infection. Figure 1 Figure 2 193 Chronic Viral Infections: HIV and Hepatitis P 165 Recurrent disease in HIV‐infected patients with Hodgkin Lymphoma (HL) or Non‐Hodgkin Lymphoma (NHL) in the German AIDS‐related lymphoma cohort study P. Schommers1, D. Gillor1, C. Wyen1, T. Wolf2, J.‐C. Wasmuth3, J. Bogner4, C. Spinner5, S. Esser6, B. Jensen7 A. Schleicher8, G. Fätkenheuer1,9, C. Hoffmann8,10 1
University of Cologne, First Department of Internal Medicine, Cologne, Germany University of Frankfurt, Department of Medicine II, Frankfurt, Germany 3
University of Bonn, Department of Internal Medicine I, Bonn, Germany 4
University of Munich, Department of Internal Medicine IV, Dept. of Infectious Diseases, Munich, Germany 5
TU Munich, Klinikum rechts der Isar, Munich, Germany 6
University of Essen, Department of Dermatology, Essen, Germany 7
University of Duesseldorf, Department of Gastroenterology, Duesseldorf, Germany 8
University of Schleswig Holstein, Campus Kiel, Kiel, Germany 9
German Centre for Infection Research (DZIF), Partner site Bonn‐Cologne, Cologne, Germany 10
ICH study centre, Hamburg, Germany 2
Objective Outcome of HIV‐infected patients (pts) with HL or NHL has been improved during recent years. However, recurrent disease (RD) after complete remission (CR) still remains a therapeutical challenge. Methods This prospective multicenter cohort study includes all HIV‐infected pts diagnosed with HL or NHL since January 2005 in 32 participating centers. This analysis focuses on pts who achieved a documented CR with first‐line conventional polychemotherapy. Results As of June 2015, 311 (235 [60%] with NHL and 76 [72%] with HL) out of 499 pts with lymphoma (394 high‐grade NHL, 105 HL) achieved a documented CR. Among these, 39 pts (8%; 31 NHL, 8 HL) had RD. Incidence of RD was 6.9/100 patient years (PY) within the first year after initial diagnosis and 1.3/100 PY thereafter (p=0.0062). Median time until RD was 7.3 months for NHL and 18.0 months for HL. Only 6 NHL RD cases occurred after 12 months, whereas all 8 HL RD cases occurred beyond 12 months (p=0.045). Median overall survival (OS) of patients with RD was 29.0 months (95% CI 14.1‐44.2 months) after initial lymphoma diagnosis and was longer in pts with HL compared to NHL (not reached vs. 15 months; p=0,024). Two‐years OS of pts with a proven CR was significantly longer for pts without a RD compared to pts with a RD (97% vs. 57%; p=<0.001)(Figure 1). The predominant cause of death in patients with RD was lymphoma (68%). Conclusions In HIV‐infected pts with lymphoma and with documented complete remission, RD rates are relatively low. However, outcome of patients with RD remains poor. While most of NHL RD cases occur within the first year after chemotherapy, most HL RD cases are diagnosed beyond 12 months. Figure 1 194 Chronic Viral Infections: HIV and Hepatitis P 166 Crispr/Cas9‐derived activation systems as novel approaches to overcome HIV latency U. Lange1,2, J. Bialek1, M. Voges1, C. Schäfer1, J. Hauber1 1
2
Heinrich Pette Institute ‐ Leibniz Institute for Experimental Virology, Hamburg, Germany University Medical Center Hamburg‐Eppendorf, Hamburg, Germany Infection with Human Immunodeficiency Virus (HIV) leads to establishment of latently infected cellular reservoirs. These reservoirs carry integrated, transcriptionally silenced provirus. Silencing hinders viral elimination by immune surveillance or therapeutic drug regimens. Therefore, latent proviral DNA is considered to be the major hurdle in HIV eradication. To overcome this hurdle, one current strategy is the use of so‐called latency reversing agents (LRAs), such as histone deacetylase inhibitors or protein kinase C pathway agonists. Through different mechanisms, these agents lead to transcriptional activation of latent provirus, making then activated reservoir cells vulnerable to immune surveillance. However, clinical performance of LRAs has so far not been convincing. One major drawback is the lack of specificity, resulting in untargeted cellular activation. We have hence explored two novel activation systems to specifically reactivate HIV long terminal repeats (LTRs) in latent cells. Both systems make use of Crispr/Cas9 technologies, allowing targeting of transcriptional activators explicitly to proviral DNA sequences. We show that particular regions within the LTR appear to be optimal for recruitment of both transcriptional activator systems. Furthermore, we demonstrate transcriptional activation of proviral genomes in a number of in culture model systems of HIV latency. In these systems, we show how observed levels of activation compare to treatment with various established LRAs. Taken together, our results present a novel, promising approach to optimize current latency reversing strategies in view of HIV elimination. 195 Chronic Viral Infections: HIV and Hepatitis P 167 Tailoring Adeno‐Associated Viral (AAV) vectors for Applications in Infection Research M. Schnödt1, C. Schäfer2, A. Nowag3, A. Huber1, U. Dietrich4, J. Hauber2, P. Hartmann3, H. Büning5 1
Zentrum für Molekulare Medizin Köln, Universität zu Köln, Cologne, Germany Heinrich‐Pette‐Institut, Hamburg, Germany 3
Klinik I für Innere Medizin, Universitätsklinikum Köln, Cologne, Germany 4
Georg‐Speyer‐Haus, Frankfurt, Germany 5
Institut für Experimentelle Hämatologie, Medizinische Hochschule Hannover, Hannover, Germany 2
Vectors based on the replication‐defective parvovirus, adeno‐associated virus (AAV), have emerged as one of the leading gene transfer systems for in vivo gene therapy. As of yet, the main focus of AAV’s clinical application is long‐term gene transfer into post‐mitotic tissues such as liver, muscle or the eye for the treatment of monogenetic diseases. Furthermore, AAV vectors do show promise as tools for transient modification of proliferating cells and as templates in gene correction approaches, and have recently entered the area of vaccine development. So far, AAV vectors have been applied in more than 120 clinical trials proofing safety for both local and systemic (by intravenous injection) administration of AAV vectors and reporting on clinical benefit for the treatment of hemophilia, Leber’s congenital amaurosis or lipoprotein lipase deficiency. However, first generation AAV vectors are based on natural isolates (serotypes) and are thus not optimized for clinical use. Especially, the presence of pre‐existing neutralizing antibodies precludes inclusion of a number of patients. Another challenge is the specific tailoring of AAV’s tropism as wild type AAV lacks cell type selectivity resulting in off‐target transduction. Moreover, distinct cell types representing important targets in gene therapy and vaccine development are not targeted in vivo and/or show low permissiveness. Since the AAV’s host interaction and its native tropism are mainly defined by the viral capsid, we develop and exploit capsid engineering to overcome these drawbacks. Efforts within the TTU HIV specifically focus on optimizing AAV‐mediated gene delivery to hematopoietic stem cells and T lymphocytes. Capsid engineering can also be exploited to augment immunogenicity of vector‐based vaccines. Combining antigen display from AAV capsids with vector‐mediated overexpression e.g. resulted in a new class of vaccines that acts as single shot prime‐boost vaccine. Based on this development, a novel strategy for inducing broadly neutralizing antibodies is currently explored as collaborative project again within the TTU HIV. These innovative techniques will be presented at the meeting. 196 Chronic Viral Infections: HIV and Hepatitis P 168 Molecular mechanisms regulating HIV‐1 latency in memory T Cells: a concerted action of PML and G9a B. Lucic1,2, C. Valentini1,2, M. Lusic1,2 1
2
University Hospital Heidelberg, Department of Infectious Diseases, Integrative Virology, Heidelberg, Germany German Center for Infection Research, Heidelberg, Germany Introduction As an obligate intracellular parasite, HIV‐1 needs to modify host cell environment to ensure its own progeny: virus fine‐tunes host‐pathogen interactions to enter the state of transcriptional latency, a main cause for failure of current antiviral therapies. We have recently reported that promyelocytic leukemia protein (PML/TRIM19) member of the TRIM family of antiviral proteins and the main organizer of PML Nuclear Bodies, cooperates with G9a/EHMT2 methyltransferase to efficiently repress viral gene expression during latency. Importantly, their removal causes loss of heterochromatin repression mark H3K9me2 and strongly activates viral gene expression in T cells (Lusic et al., 2013). Objectives To further analyze nuclear regulatory mechanisms of T cells upon HIV‐1 infection, we aim at determining the contribution of PML and G9a to the establishment of latency. At the same time we want to explore their role in the general genome organization of T cells: considering the stochastic nature of gene expression and hence reactivation from HIV‐1 latent state, we will assess the role of these proteins in cellular gene expression control at the single cell level. Material and methods We used RNA‐Seq and quantitative PCR to assess expression levels of PML and G9a genes. For the generation of PML and G9a KD cells we used lentiviral and adeno‐associated delivery systems. Isolated primary CD4+ cells and their Memory and Naïve subsets were sorted using BD FACSVerse instrument and single cell experiments were performed on Fluidigm C1 instrument. Results To explore the role of PML and G9a in establishment and maintenance of latency we treated primary CD4+ cells with arsenic III oxide‐ known to downregulate PML levels as well as with BIXO1294, G9a inhibitor. Cells were subsequently infected with HIV‐1 and left to enter latency. Ex‐vivo latency models established in the conditions of decreased PML levels showed that the virus continues to replicate as opposed to the conditions of normal PML levels, where is gradually silenced. In our preliminary RNA‐Seq data performed on a population of lymphoid cells we observed that a particular sets of human genes are differentially expressed and spatially distributed in the PML depleted conditions. We want to expand these findings on G9a with a goal to further understand how these two factors act together in CD4+ memory T cells and macrophages, major reservoirs of latent virus. The role of these proteins in transcription is also explored at the single cell level; the first single cell experiment is performed, and the data are currently analyzed. Conclusion We identify PML and G9a as human gene regulation modifiers during latency. Characterizing molecular mechanisms of their action has a great potential for development of future strategies to eliminate the latent viral reservoir and cure the disease. 197 Chronic Viral Infections: HIV and Hepatitis P 169 Peripheral nuclear localization of HIV‐1 and a missing link to proviral latency: role for Tpr protein C. Valentini1,2, B. Lucic1,2, M. Stanic1,2, M. Lusic1,2 1
2
University Hospital Heidelberg , Department of Infectious Diseases, Integrative Virology, Heidelberg, Germany German Center for Infection Research, Heidelberg, Germany Introduction A remarkable feature of HIV‐1 latency is the persistence of a stably integrated virus in a transcriptionally silent state. Important roles in the establishment of latency are played by genomic localization of integration sites, epigenetic regulation of viral transcription and dependence of HIV‐1 transcriptional status on nuclear topology. HIV‐1 preferentially integrates into active genes and exploits cellular mechanisms to control its own gene expression by either efficiently replicating its genome or undergoing transcriptional silencing. Recently, we demonstrated that highly targeted genes, named Recurrent Integration Genes (RIGs), are non‐randomly distributed within the nucleus. By 3D Immuno DNA FISH (Fluorescence In Situ Hybridization) in activated primary CD4+ cells we found that RIGs localize preferentially in the outer shells of the nucleus. Consistently, the newly integrated provirus is found at the nuclear periphery in primary CD4+ T‐cells, avoiding the inner part of the nucleus as well as Lamin Associated Domains at the periphery. Furthermore, by chromatin immunoprecipitation on primary infected CD4+ T‐cells, we found that active HIV‐1 genomic sequences bound by RNA Polymerase II are also bound by nucleoporins of the inner basket of Nuclear Pore Complex (NPC). Objectives Unraveling the role of Tpr in peripheral positioning of the provirus, in transcriptional control and latency, and in maintaining chromatin features at the nuclear periphery. Materials & Methods T cell lines and primary CD4+ cells are treated with siRNA or AAV‐delivered shRNA, infected with pseudotyped pNL4.3‐EnvFS and harvested for 3D‐ImmunoDNA FISH and CHIP analysis. Results In TPR depleted T cells, HIV‐1 integration efficiency is not impaired, but 3D Immuno DNA FISH analysis reveal that the provirus is localized toward the center of the nucleus. Both in a cellular and in a primary model of latency, HIV‐1 genome retains its peripheral localization. However, when localization is analyzed at molecular resolution by ChIP using antibodies against nucleoporins, binding of the proviral region to the NPC is observed upon transcriptional activation but not in latent conditions. Nucleoporins directly participate in HIV‐1 transcriptional regulation, since proviral transcription is significantly reduced when Tpr and Nup153 are silenced. Conclusions Taken together, these findings underline the importance of nuclear topology and the role of nucleoporins in regulating transcription of HIV‐1 and possibly, establishment of latency. Unravelling how T cells regulate their gene expression patterns during latency and the role of Tpr in conditioning proviral transcriptional fate at the nuclear periphery will provide new venues for viral eradication. 198 Chronic Viral Infections: HIV and Hepatitis P 170 Dissecting the influence of HIV infection and treatment with antiretroviral therapy on Human Papillomavirus infection, disease and immunity T. Lennemann1,2, R. Mcharo1, L. Torres3, A. Bauer1,2, M. Chachage1, D. Kowuor1, D. Hoefler4, R. Koup5, J. Mnkai1 M. Mwakatima1, N. Mwinuka1, M. Pawlita4, N. Schroeder6, M. Hoelscher2,7, L. Maboko1, F. Rwegoshora3 C. Geldmacher2,7, A. Kroidl2,7 1
NIMR‐Mbeya Medical Research Center, Mbeya, Tanzania Medical Center of the University of Munich (LMU), Division for Infectious Diseases and Tropical Medicine, Munich, Germany 3
Mbeya Referral Hospital (MRH), Mbeya, Tanzania 4
German Cancer Research Center (DKFZ), 4Division Molecular Diagnostics of Oncogenic Infections, Infection, Inflammation and Cancer Program, Heidelberg, Germany 5
Vaccine Research Center (NIH/NIAID), Bethesda, United States 6
University of Magdeburg, Institute for Pathology, Magdeburg, Germany 7
German Center for Infection Research (DZIF), partner site Munich, Munich, Germany 2
Background Cervical Cancer (CC) is the most frequent cancer in women in Sub Saharan Africa, where HIV is co‐prevalent. HIV infection is associated with a substantially increased risk for CC even in the era of antiretroviral therapy (ART). The ongoing 2H study aims to dissect the influence of HIV infection and ART on Human papilloma virus (HPV) infection, disease and immunity to better understand HIV‐HPV co‐pathogenesis and improve prevention and therapeutic intervention strategies. Materials and Methods HIV+ and HIV‐ Women attending the national Cervical Cancer screening program in Mbeya, Tanzania, are screened for cervical intraepithelial lesions and CC using pap smear and using biopsy and histology for verification. CD4 counts, quantitative HIV‐RNA and data on ART are collected for HIV+ women. Cases (CC and High Grade Intraepithelial Lesions (HSIL, includes CIN II/III)) and controls (no lesion and Low Grade Intraepithelial Lesion (LSIL, CIN I)) are studied in more detail to identify cervical HPV genotypes and ‐ using an IFNg ELISPOT assay ‐ to characterize T cell responses to oncogenes of high risk (hr) HPV types in relation to HPV disease, HIV and ART status. Results As of May 2015, 511 HIV+ and 716 HIV‐ women were screened. HIV infection was associated with an increased frequency of LSIL (9.5% versus 1.1%, risk ratio 8.4) and HSIL (3.3% versus 1.1%, risk ratio 3.0). The frequency of CC was comparable between HIV+ and HIV‐ women (2.5% versus 3.7%). The median age of HIV+ women with CC was 12 years younger (43 years, p=0.008) compared to HIV‐ women. 60% and 80% of HIV+ women with CC and HSIL, respectively, were on ART. Frequent hrHPV types detected in HIV+ (n=12) and HIV‐ CC cases (n=16) were HPV16 (67% and 31%, respectively), HPV45 (17% and 38%) and HPV18 (31.3% and 17%). HIV+ women had a 1.7‐fold increased risk of an infection with HR‐HPV (68.4% versus 40.2%) and were more often infected with multiple hrHPV types (mean: 1.3 versus 0.6). Fewer hrHPV types were detected in HIV+ CC cases (mean: 1.21, n=12 cases) compared to HIV+HSIL+ (mean: 2.2, n=12 cases) and to HIV+LSIL+ cases (mean: 1.9, n=42 cases). Of note, hrHPV type prevalence differed between HSIL and CC cases; a HPV16 single infection was detected in 60% of HIV+ CC cases, whereas more diverse hrHPV types (lead by HPV35: 55.5%) were detected in HIV+ HSIL cases. E6‐ and E7‐specific T cell responses for the clinically most relevant HR HPV types 16, 18, 45 and 35 were selectively depleted in HIV+ women with CC or HSIL compared to women without lesions, (combined p‐
value=0.016 (E6), p‐value=0.0014 (E7)) Conclusion Despite ART, HIV infection is linked to accelerated HPV disease progression. Although a remarkable diversity of hrHPV types was detected in HIV+ women with no lesion, LSIL or HSIL ‐ a HPV16 single infection was detected in the majority of CC cases. hrHPV genotyping could potentially help to guide therapeutic intervention in HIV+ women with LSIL or HSIL. 199 Chronic Viral Infections: HIV and Hepatitis P 171 CD25+FoxP3+ memory CD4 T cells are frequent targets of HIV infection in vivo M. Chachage1, G. Pollakis2, E. O. Kuffour3,4, K. Haase5, A. Bauer1,6, Y. Nadai6, L. Podola1,6, P. Clowes1,6 M. Schiemann7,8, L. Henkel7, D. Hoffmann9, S. Josephs10, S. Bhuju11, L. Maboko1, F. S. Sarfo12, K. Eberhardt3 M. Hoelscher13,14, T. Feldt3,4, E. Saathoff13,14, C. Geldmacher13,14 1
NIMR‐Mbeya Medical Research Center, Mbeya, Tanzania University of Liverpool, Institute Institute of Infection and Global Health, Liverpool, Great Britain 3
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany 4
University Hospital Düsseldorf, Department of Gastroenterology, Hepatology and Infectious Diseases, Duesseldorf, Germany 5
Technische Universität München, Department of Genome Oriented Bioinformatics, Munich, Germany 6
Klinikum der Universität München, Abteilung für Infektions‐ und Tropenmedizin, Munich, Germany 7
Technische Universität München, Institute for Medical Microbiology, Immunology and Hygiene, Munich, Germany 8
Technische Universität München, “Antigen‐specific Immunotherapy” and “Immune‐Monitoring”, Helmholtz Center Munich (Neuherberg), Munich, Germany 9
Technische Universität München, Institute for Virology, Munich, Germany 10
MRC Clinical Trials Unit at UCL, London, Great Britain) 11
Helmholtz Centre for Infection Research, Genome Analytics, Braunschweig, Germany 12
Komfo Anokye Teaching Hospital, Kumasi, Ghana) 13
Medical Center of the University of Munich (LMU), Division for Infectious Diseases and Tropical Medicine, Munich, Germany 14
German Center for Infection Research (DZIF), partner site Munich, Munich, Germany 2
Background Interleukin 2 (IL2) signaling induces homeostatic proliferation and maintenance of IL2 receptor alpha chain+ (CD25) FoxP3+CD4+ T cells in vivo and facilitates HIV replication in vitro. We therefore wanted to address whether CD25+FoxP3+CD4+ T cells constitute a suitable cell subset for HIV infection and plasma virus production in vivo. Materials & Methods CD25+FoxP3+CD4+ T cell frequencies, absolute numbers and the expression of CCR5 and cell cycle marker Ki67 were studied ex vivo in peripheral blood cells from HIV+ and HIV‐ study volunteers. Different memory CD4 T cell subsets, including CD25+FoxP3+ memory CD4+ T cells, were then sorted to quantify cell‐associated HIV‐
DNA using real time PCR and to study the phylogenetic relationship between cell‐associated and plasma sequences for the highly variable HIV_EnvV1V3 region (Hxb 6559 ‐ 7320). Results A substantial fraction of CD25+FoxP3+CD4+ T cells (median >50%) expressed the HIV co‐receptor CCR5 and high frequencies of Ki67+ CD25+FoxP3+CD4+ T cells were observed in HIV+ subjects (median: 27.6%). HIV‐DNA content was 15‐fold higher in CD25+FoxP3+CD45RO+ CD4+ T cell compared to CD25‐FoxP3‐CD45RO+CD4+ T cells (p=0.0032). Phylogenetic distance between different cell associated and plasma virus EnvV1V3 sequences significantly increased with HIV infection duration (p<0.05) regardless of the memory CD4+ T cell subset. Conclusions Our data demonstrate high in vivo HIV infection rates of circulating CD25+FoxP3+CD45RO+CD4+ T cells, probably linked to CCR5 expression and high in vivo proliferation of this cell subset. However our data argue against a substantial contribution of any circulating memory CD4+ T cell subsets to plasma virus production. 200 Chronic Viral Infections: HIV and Hepatitis P 172 The late presenting HIV‐infected patient 30 years after introduction of HIV testing: Spectrum of opportunistic diseases and “missed opportunities” to diagnose HIV‐infection D. Tominski1, J. Katchanov1, D. Driesch2, K. Arasteh1, H. Slevogt1,2, H. Stocker1 1
2
Auguste Viktoria Klinikum, Infectious Diseases, Berlin, Germany Universität Jena, Jena, Germany Background In Europe approximately 30% of individuals are not diagnosed as HIV‐infected until late in the course of their disease. The proportion of these patients who seek medical care either with HIV‐indicator conditions or with AIDS before their HIV‐infection is detected is unknown. Objectives To describe the characteristics of HIV late presenters and their opportunistic diseases at diagnosis. To determine the proportion of individuals that seek medical care with HIV‐indicator conditions or AIDS prior to HIV diagnosis and to define the risk factors for not being tested despite the presence clear indicators of HIV‐
infection. Methods Single centre, retrospective cohort study. Results In the 5 year period of 2009‐2013 268 “late presenters” were identified and stratified into two groups. Group A were patients without documented HIV‐indicators. Group B had documented HIV‐indicators at visits prior to HIV diagnosis. The most common AIDS defining conditions among the total study population was oesophageal candidiasis (n=136; 50.7%) followed by wasting syndrome (n=106; 39.6%) and pneumocystis pneumonia (n=91, 34.0%). 55 patients (20.5%) had at least one HIV indicator condition or AIDS upon preceeding contacts with health care services without being offered an HIV test. Women and non‐MSM patients had significantly higher odds of being amongst patients with indicators without being tested (OR 4.7, 95%CI 2.2‐10.0, p Most common “missed” indicator conditions were leukocytopenia (n=13, 23.6%), thrombocytopenia (n=12, 21.8%), oral candidiasis (n=9, 16.4%), unexplained weight loss (n=7, 12.7%), H. zoster (n=5, 9.1%) and ‐in female patients‐ cervical dysplasia or cancer (n=4, 20% of women). The median time between the presentation with an indicator condition and the establishment of HIV‐diagnosis was 158.5 days (IQR: 40‐ 572). Patients with oral candidiasis and unexplained weight loss had the shortest time between the missed opportunity and the establishment of HIV infection.Fifty five hospital admissions with overall in‐patient care cost of over 500 000 Euro and ‐most importantly‐ 6 in‐hospital deaths might have been prevented if HIV testing would have been done in patients with documented indicator conditions. Conclusions Indicator conditions are still missed by clinicians. Women and non‐MSM are at highest risk of missed opportunities. 201 Chronic Viral Infections: HIV and Hepatitis P 173 Characterization of innate lymphoid cells (ILCs) in the human gastrointestinal tract demonstrates compartment‐specific ILC distribution which is markedly altered in HIV infection F. Goeser1,2, B. Krämer1,2, P. Lutz1,2, A. Glässner1,2, F. Wolter1,2, D. Kaczmarek1,2, P. Kokordelis1,2, C. Boesecke1,2 C. Schwarze‐Zander1,2, T. Weismüller1,2, C. P. Strassburg1,2, J. Rockstroh1,2, U. Spengler1,2, J. Nattermann1,2 1
2
Universitätsklinikum Bonn, Medizinische Klinik und Poliklinik I, Bonn, Germany Deutsches Zentrum für Infektionsforschung, Bonn, Germany Background Innate lymphoid cells (ILC) have been shown to display critical effector and regulatory functions in innate immunity and tissue remodelling. Recent reports demonstrate ILCs to promote anatomical containment of gut commensal bacteria and to play an important role in intestinal inflammation. However, most of these studies have been performed in mouse models and, thus, the distribution of ILCs in the human gastrointestinal (GI) tract remained unclear. Methods In HIV(‐) individuals biopsies from macroscopically and histologically normal tissue were taken in the oesophagus (n=8), the stomach (n=11), the duodenum (n=11), the terminal ileum (n=13), and the left‐sided colon (n=13) during routine endoscopy. Moreover, oral biopsies (n=6) as well as peripheral blood (n=14) was studied. In addition, biopsies from the oesophagus (n=7), the stomach (n=12), the duodenum (n=12), the terminal ileum (n=8), and the colon (n=8), as well as PBMC (n=14) from HIV‐infected patientsunder effective therapy were analysed. ILCs were phenotypically characterized by flowcytometry and tested for expression of IFN‐g, IL‐13, and IL‐22 following stimulation with PMA and ionomycin. Results Frequency of total ILC (as % of Lin(‐) cells) stepwise increased from the oral cavity to the distal GI‐tract, with highest frequencies found in the colon. The only exception was the oesophagus displaying an ILC‐frequency close to that observed in the ileum. Frequency of CD127(+)ILC1 was significantly higher in upper GI‐tract (mouth, stomach, duodenum) than in ileum and colon. Similar observations were made regarding the distribution of CD103(+)ILC1. The opposite was true regarding ILC3, which represented the majority of ILCs in the ileum and the colon but only a minor fraction in the oesophagus and stomach, respectively. Substantial proportions of ILC2 were only found in the stomach and the oral cavity. Expression of IFN‐g(ILC1), IL‐13(ILC2), and IL‐22(ILC3) did not differ significantly between the analysed GI‐compartments. Distribution of total ILCs in HIV(+) patients was similar to that observed in healthy controls. However, HIV infection was associated with a dramatic decrease in CD127(+)ILC1 in all analysed parts of the GI tract. In contrast, we found the frequency of CD103(+)ILC1 to be significantly higher in the proximal part of the intestine in HIV infection than in healthy controls. Conclusions Our data indicate a compartment‐specific distribution of ILCs in the human GI‐tract, which is markedly dys‐
regulated in HIV infection. 202 Chronic Viral Infections: HIV and Hepatitis P 174 HIV mono‐infection is associated with an impaired anti‐HCV activity of NK cells F. Goeser1,2, B. Krämer1,2, A. Glässner1,2, P. Kokordelis1,2, C. Boesecke1,2, F. Wolter1,2, C. Schwarze‐Zander1,2 D. Kaczmarek1,2, U. Spengler1,2, J. Rockstroh1,2, J. Nattermann1,2 1
2
Universitätsklinikum Bonn, Medizinische Klinik und Poliklinik I, Bonn, Germany Deutsches Zentrum für Infektionsforschung, Bonn, Germany Background HCV infection in HIV(+) patients is associated with faster liver disease progression compared to HCV mono‐
infection. HIV‐associated immune defects are considered to play an important role in this context. Here, we analysed the effects of HIV‐infection onNK cell anti‐HCV. Methods 28 HIV‐infected patients, including 20 individuals that were HIV RNA(‐) under HAART and 8 treatment‐naïve HIV RNA(+) patients, as well as 11 healthy HIV(‐)/HCV(‐) controls were enrolled into this study. NK cell‐mediated inhibition of HCV replication was analyzed using the HuH7A2HCVreplicon model. NKp46 expression and IFN‐g production of NK cells as well as IL‐2 secretion of CD4+ T lymphocytes were studied by flow cytometry. Results PBMC from HIV(+) patients displayed a significantly impaired anti‐HCV activity, irrespective of cART, which could in part be explained by HIV‐associated decline in NK cell numbers. In addition, NK cell IFN‐g production was significantly impaired in HIV infection. Of note, low frequency of IFN‐g(+) NK cells in HIV(+) patients was associated with ineffective inhibition of HCV replication. Inhibition of HCV replication as well as IFN‐g production of NK cells was positively correlated with CD4+ T cell counts in untreated HIV RNA(+) patients but not in those under effective cART. Moreover, we found viraemic HIV infection to be associated with reduced IL2 production of CD4+ T cells compared to healthy controls and HIV RNA(‐) patients. In line with previous results of our group we found activated CD4+ T cells to effectively stimulate IFN‐g production of NK cell from healthy controls. Surprisingly, the ability of CD4+ T cells to trigger activity of HIV(‐) NK cell did not differ significantly between controls and HIV(+) patients. However, when NK cells from HIV(+) patients were tested we found that neither autologous CD4+ T cells nor cells from healthy controls were able to trigger IFN‐g production, indicating an NK cell intrinsic defect. Accordingly, we found HIV infection to be associated with an impaired NK cell response to stimulation with recombinant IL‐2. Conclusions HIV infection per se has a strong suppressive effect on anti‐HCV activity of NK cells. This may contribute to low spontaneous clearance rate and accelerated progression of HCV‐associated liver disease observed in HIV(+) patients. 203 Chronic Viral Infections: HIV and Hepatitis P 175 Influence of HLA glycosylation on the binding of HLA‐B*57:01 to KIR3DL1 W. Salzberger1, W. Garcia‐Beltran2, H. Dugan2, S. Gubbala2, C. Simoneau2, S. Gressens2, S. Jost2, M. Altfeld1,2 1
2
Heinrich‐Pette‐Institut, Virus Immunologie, Hamburg, Germany Ragon Institute of MGH, MIT, and Harvard, Cambridge, USA Background The NK cell receptors and their ligands have been shown to play an important role in determining human immunodeficiency virus‐1 (HIV‐1) disease progression, one of the most notable cases being high‐expressing KIR3DL1 allotypes in combination with HLA‐B*57:01, which correlate with elevated NK cell effector function. A number of viral and bacterial infections, including HIV‐1, have been shown to affect N‐glycans on IgG, which are structurally related to N‐glycans on HLA class I proteins. Given the importance of glycosylation on protein‐protein interactions and the ability of infections to affect the glycosylation of host proteins, we were interested in the effect that modulation of HLA‐B*57:01 glycosylation had on the KIR:HLA binding and its effect on NK cell function. Methods 721.221 cells transduced with different HLA class I molecules were treated with different glycosylation enzyme inhibitors. HLA expression was measured with pan‐ and specific HLA class I antibodies and KIR binding was measured using KIR‐Fc fusion constructs. HLA‐B*57:01‐transduced 721.221 cells were deglycosylated using tunicamycin, and consequences on KIR3DL1 binding was determined using primary KIR3DL1+ NK cells and Jurkat cells transduced with a KIR3DL1‐CD3ζ chimeric receptor. Results The different glycosylation enzyme inhibitors had varying effects on the expression and KIR‐Fc binding of different HLA class I proteins. Inhibition of the first step of N‐glycosylation by tunicamycin resulted in the overall maintenance of HLA‐B*57:01 expression on 721.221 cells, but binding of KIR3DL1‐Fc to 721.221 cells was severely decreased. This effect of tunicamycin on KIR3DL1 binding was corroborated by functional data in both KIR3DL1ζ+ Jurkat reporter cells as well as KIR3DL1+ NK cells. Conclusions While much of the role of modified glycosylation patterns remains to be elucidated, our results demonstrate the importance of glycosylation for the HLA‐KIR interaction and NK cell activation, which might have implications in NK cell recognition of infected target cells. 204 Chronic Viral Infections: HIV and Hepatitis P 176 Assessment of early kinetics of sequential gag mRNA and protein expression following HIV‐
1 latency reversal using flow‐based techniques G. Martrus1, A. Niehrs1, M. Altfeld1 1
Heinrich Pette Institut, Hamburg, Germany Background Upon infection, HIV‐1 establishes a pool of persisting latently infected cells. Although antiretroviral treatment can efficiently target HIV‐1 replication, it cannot eradicate the established reservoir. New drug therapies are focusing on reactivating the latently infected cells. Nevertheless, it remains uncertain to what extent latently infected cells are reactivated. We established a new technique to evaluate the molecular kinetics of HIV‐1 latency reactivation on the single cell level, distinguishing cells in which only viral mRNA is expressed from cells in which viral proteins are produced. Methods J89 cells were used as an HIV‐1 latency reactivation model. Cells were treated with different concentrations of hTNFα cytokine and an HDAC inhibitor, Romidepsin (RMD), for defined time periods. Using a newly established technique that allows for simultaneous detection of mRNA targets and intracellular proteins by multiparameter flow cytometry, combined intracellular staining for Gag p24 protein and Gag p24 mRNA was performed. Results Following stimulation with 10ng/ml of hTNFa or 5nM RMD for 24hrs, three distinct cell populations were distinguishable: single‐positive p24 mRNA+ cells, double‐positive p24 mRNA/protein+ cells and double‐negative cells. p24mRNA+ cells were detected 3hrs post‐stimulation (hTNFa) and 6hrs post‐stimulation (RMD) in the absence of intracellular Gag protein detection. With increasing time, the single‐positive p24mRNA+ population shifted towards a double‐positive p24 mRNA+/protein+ population. Conclusion Using a novel flow cytometry based method, the kinetics of HIV‐1 mRNA and protein synthesis upon latency reactivation were determined, and the consequences for the expression of specific surface markers that might enable immune recognition of HIV‐1‐infected cells. 205 Chronic Viral Infections: HIV and Hepatitis P 177 Parallel assessment of Th17 cell frequencies by surface marker co‐expression versus ex vivo IL‐17 production in HIV‐1 infection. G. Dunay1, I. Tóth2, J. Eberhard1,3, O. Degen3, E. Tolosa4, J. van Lunzen1,3, J. Hauber1, J. Schulze zur Wiesch1,3 1
Heinrich‐Pette‐Institut Leibniz‐Institut für Experimentelle Virologie, Abteilung Antivirale Strategien, Hamburg, Germany National Institutes of Health, National Institute of Allergy and Infectious Diseases, Vaccine Research Center, Human Immunology Section, Bethesda, USA 3
Universitätsklinikum Hamburg‐Eppendorf, I. Med., Abteilung Infektiologie, Hamburg, Germany 4
Universitätsklinikum Eppendorf, Institut für Immunologie, Hamburg, Germany 2
Introduction Th17 cells can either be identified by intracellular cytokine staining (ICS) for IL‐17 production or by co‐staining of surface markers. Discrepancies regarding the published frequencies of Th17 cells in peripheral blood mononuclear cells (PBMC) of HIV patients may partly be due to the different methodologies used. So far, a parallel comparison of the two methods has not yet been carried out in the context of HIV‐1 infection. Methods Cryopreserved PBMCs from healthy controls and HIV infected subjects including treated (cART) and viremic patients were split and analyzed side by side by flow cytometry for expression of surface markers CCR6, CXCR3, CCR4 and CD161, or for intracellular expression of IL‐17A and IFNγ after stimulation. Results The frequencies delivered by the two methods significantly correlated, however, the characterization of Th17 cells as CCR6+CXCR3‐CCR4+CD161+ yielded considerably higher frequencies than the corresponding frequencies obtained by characterization via cytokines (IL‐17+IFNγ‐), regardless of the HIV status. The relative frequency of Th17 cells within the CD4+ compartment was preserved in HIV infection, but there was a significant decrease in the absolute Th17 number, which was restored after initiation of cART, paralleling CD4+ restoration. Absolute Th17 numbers were inversely correlated with HIV viral loads. Conclusion The definition of Th17 cells by surface markers might overestimate their frequency in comparison to functional assessment of IL‐17 production by ICS. However, both methods yield proportionate results with reduced absolute numbers of Th17 cells in untreated HIV disease, reflecting the depletion of total CD4+ T cells in viremic HIV patients, and restoration with cART. 206 Chronic Viral Infections: HIV and Hepatitis P 178 MAIT cell frequencies are severely reduced in HIV‐1/HCV co‐infection and HIV‐1 but not HCV mono‐infection J. Eberhard1, S. Kummer1, P. Dierks1, C. Ackermann1, C. Scheurich1, J. Schulze zur Wiesch1 1
University Medical Center Hamburg‐Eppendorf, Hamburg, Germany An early and irreversible loss of MAIT cells has been shown in HIV infection. This loss has been explained by increased recruitment and increased death of MAIT cells as well as exhaustion and down‐regulation of the surface marker CD161. A loss of MAIT cells might contribute to the loss of integrity of the intestinal barrier and increased microbial translocation in HIV, HCV and HIV/HCV Co‐infection. MAIT cells seem to play an important role in the liver as well. However, a description of MAIT cell frequencies in HIV, HCV or HIV/HCV Co‐infection are missing so far. Here, we present data comparing CD161+ MAIT cell frequencies in a cohort comprising HCV ( n= 20) and HIV‐1 (n=12) mono‐infected patients, HIV‐1/HCV co‐infected (n=12) patients and healthy controls (n=21). Both patient groups infected with HIV‐1 received antiretroviral treatment. While we observed a slight but non ‐
significant reduction of MAIT cells in HCV infection we saw a profound depletion in ART treated HIV‐1 monoinfected and ART treated HIV‐1/HCV coinfected patients. In these groups the remaining MAIT cells were highly activated evaluated by the expression of CD38 in combination with HLA‐DR. In conclusion microbial translocation and global lack of MAIT cells may be a fundamental mechanism through which HIV accelerates progression of chronic liver disease. 207 Chronic Viral Infections: HIV and Hepatitis P 179 Increased Frequency of CD49b/LAG‐3+ Type 1 regulatory T cells in the GUT of HIV infected Individuals J. Koch1, G. Fätkenheuer1, C. Lehmann1 1
Universitätsklinikum Köln, Klinik I für Innere Medizin, Cologne, Germany Background Elevated serum levels of interferon alpha (IFN) produced by plasmacytoid dendritic cells (pDC), and Interleukin‐10 (IL‐10) are associated with immune hyperactivation and disease progression. Recently, coexpression of CD49b and LAG‐3 was shown to identify Tr1 cells, which secrete large amounts of the immunesuppressive cytokine IL‐10. Moreover, the gut associated lymphatic tissue (GALT) represents the largest lymphoid organ. Local immune responses lead to changes in mucosal integrity and there is evidence that these local events play a central role in HIV‐ immunopathogenesis. However, despite the importance of the gut mucosae for the immune activation in HIV pathogenesis little is known about pDC and Tr1‐cells in these tissues during chronic infection. Methods We analyzed pDC (BDCA2+CD123+), Tr1‐cells (CD3+CD4+CD49b+LAG‐3+) and ‚bystander’ CD4+ and CD8+ T‐
cells (CD8+HLA‐DR+CD38*; CD4+HLA‐DR+CD38*) from the terminal ileum and peripheral blood by flow cytometry in treated HIV‐infected individuals (n=12) and healthy subjects (n=6). Mucosal mononuclear cells were enzymatically and mechanically isolated. Molecular analyses of Il‐10, MxA mRNA (effector molecule of IFN‐α), TLR‐7 and IFN‐y in peripheral blood and whole ileum‐biopsy pieces were performed by RT‐PCR. Data were analyzed using Mann Whitney, Wilcoxon matched pairs test and Spearman rank tests as applicable. Results HIV‐infected individuals showed significantly higher frequency of pDC and Tr1‐cells of all mononuclear cells in the GALT as compared to peripheral blood (pDC: p=0.007; Tr1: p= 0.03) and controls (pDC: p=0.01; Tr1: p=0.02). CD49b/LAG‐3+ Tr1 cell frequency was increased in the gut despite CD4+ T cell loss. Whole biopsy quantification showed elevated levels of Il‐10, MxA and TLR7 mRNA transcripts in the GALT of HIV infected patients, which positively correlated with the expression of CD38 and HLA‐DR on CD8+ T‐ cells in the GALT (r=‐0.06, p=0.008). Conclusions This is the first study reporting that the frequency of the recently described CD49b/LAG‐3+ Tr1 cells increase in the gut of HIV infected patients. While pDC accumulation in the mucosa could help to control HIV replication, over‐production of IFN‐α may also contribute to increase the immune activation. Finally, the high CD49b/LAG‐
3+Tr1 cell frequency observed in these patients, supports the idea of thereby counteracting an overly intense immune response. 208 Chronic Viral Infections: HIV and Hepatitis P 180 Untersuchungen zur Prävalenz von Humanen Papillomviren bei Schwangeren im ländlichen Ghana M. H. Schulze1, F. Völker1, R. Lugert1, P. Cooper2, U. Groß1, H. Pfister3, S. Silling3 1
Universitätsmedizin Göttingen, Institut für Medizinische Mikrobiologie, Göttingen International Health Network, Göttingen, Germany 2
St. Martin de Porres Hospital, Eikwe, Ghana 3
Institut für Virologie, Nationales Referenzzentrum für Papillom‐ und Polyomaviren, Uniklinik Köln, Cologne, Germany Fragestellung Humane Papillomviren (HPV) sind die Hauptursache für die Entstehung von Gebärmutterhalskrebs. Mit Hilfe der zur Verfügung stehen Impfstoffe (ein 2‐valenter gegen HPV16 und 18; ein 4‐valenter gegen HPV6, 11, 16 und 18; ein 9‐valenter gegen HPV6, 11, 16, 18, 31, 33, 45, 52 und 58) steht eine hocheffektive Prävention zur Verfügung, Gebärmutterhals zu verhindern. Epidemiologische Daten, die zur Entwicklung der Impfstoffe geführt haben, stammen vorwiegend aus Industrienationen. Das größte Krankheitsaufkommen an Gebärmutterhalskrebs findet sich jedoch in Sub‐Sahara Afrika. Um bei der Entwicklung zukünftiger HPV‐
Impfstoff‐Generationen eine bessere Zusammensetzung der im Impfstoff enthaltenen Genotypen zu gewährleisten, ist es unerlässlich die HPV‐Epidemiologie bisher vernachlässigter Regionen zu kennen und zu berücksichtigen. Methoden In einem Dreimonatszeitraum wurden in einem Krankenhaus im ländlichen Ghana kurz vor der Entbindung stehende Schwangere im Rahmen ihrer regulären Schwangerschaftsfürsorge mit Hilfe eines Routine‐
Vaginalabstriches auf eine Infektion mit Humanen Papilloma Viren untersucht. Entsprechend der IARC‐
Klassifikation von 2012 erfolgte das HPV‐Screening für 25 Hochrisiko‐Typen, HR, (HPV16, 18, 26, 30, 31, 33, 34, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, 85, und 97) und 19 Niedrigrisiko‐Typen, LR, (HPV6, 11, 27, 40, 42, 43, 44, 54, 55, 57, 61, 71, 72, 74, 81, 83, 84, 89, und 177) (1). Ergebnisse Bei 55 von 165 untersuchten Schwangeren (33,3%) wurde eine HPV‐Infektion festgestellt [95 % Konfidenzinterval 26.2% ‐ 41.1%]. Für 146 (49 HPV positive und 97 HPV negative) von 165 Schwangeren (88,5%) lagen Angaben zum Alter vor: Median 26 Jahre; Range 14‐49 Jahre, Mean 26,3 Jahre). Die Prävalenz der fünf häufigsten gefunden HPV‐Typen war: LR HPV6 mit 4.8%, HR HPV52 und 67 bzw. LR HPV61 mit je 4.2%, HR HPV 53 bzw. LR HPV 54 mit je 3.6%. Die fünf häufigsten HR HPV‐Typen waren: HPV52 und 67 mit je 4.2%, HPV53 mit 3.6%, HPV45 mit 3.0% und HPV18 mit 2.4%. Für HR HPV16 wurde eine Prävalenz von 1,2% ermittelt. Schlussfolgerungen Die aktuell zugelassenen Impfstoffe gegen HPV berücksichtigen die in dieser Untersuchung am häufigsten gefundenen Hochrisiko‐HPV Typen nicht in ausreichendem Maße. Um zukünftig bei der Entwicklung neuer HPV‐Impfstoff‐Generationen verstärkt die HPV‐Epidemiologie in Sub‐Sahara Afrika einschließen zu können, sollten verstärkt Untersuchungen in bisher unterrepräsentierten Regionen durchgeführt werden. Literatur 1. IARC. Biological agents. Volume 100 B. A review of human carcinogens. IARC Monogr Eval Carcinog Risks Hum. 2012/11/30 ed; 2012:1‐441. 209 Chronic Viral Infections: HIV and Hepatitis P 181 Generation of cell lines that support the full lifecycle of Hepatitis Delta Virus. F. A. Lempp1, L. Rieble1, S. Urban1,2 1
2
Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany German Center for Infection Research (DZIF), partner site Heidelberg, Heidelberg, Germany Chronic infection with Hepatitis Delta Virus (HDV) is considered the most severe form of viral hepatitis, frequently leading to the development of cirrhosis and hepatocellular carcinoma. HDV is a defective RNA virus that depends on the Hepatitis B Virus envelope proteins for progeny virus secretion and spread. With the identification of sodium‐taurocholate cotransporting polypeptide (NTCP) as bona fide receptor for both viruses, an important cellular restriction factor for viral entry has been elucidated. Cell lines stably expressing human NTCP are susceptible to HDV, replicate RNA and express Hepatitis Delta Antigen (HDAg), however, virus assembly is blocked due to the lack of HBV surface proteins. In order to establish a cell culture model that supports the full HDV viral lifecycle, we stably transduced HuH7 or HepG2 cells with both NTCP and the HBV surface proteins. Efficient expression of all transgenes was shown by taurocholate uptake assay and the analysis of HBsAg secretion in the culture supernatant. When infected with HDV, the cells secrete infectious progeny virions for more than 20 days, as shown by using an infectivity assay of the cell supernatant. This novel cell culture model can be used for various different purposes: (I) it will allow to study the complete viral lifecycle in more detail; (II) it can be used to measure replication efficiency of different HDV genotypes; (III) it can be used to analyze quasispecies generated during replication/propagation. Most important, the cell line can be used for screening purposes to find drugs that interfere with early but also late steps of the viral lifecycle or to evaluate novel drugs that are currently being developed. 210 Chronic Viral Infections: HIV and Hepatitis P 182 Selective digestion of HBV rcDNA by T5 exonuclease allows reliable PCR‐based quantification of cccDNA in in vitro infection systems B. Qu1, Y. Ni1, F. A. Lempp1, S. Urban1,2 1
2
Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany German Center for Infection Research (DZIF), partner site Heidelberg, Heidelberg, Germany In chronic HBV infection, a persistence reservoir of covalently closed circular DNA (cccDNA) serves as a transcriptional template and represents an attractive target for future curative therapies. Sensitive quantification of cccDNA requires specific PCR‐based detection methods. Specificity can be achieved through the design of primers spanning nick and gap regions that discriminate cccDNA from the excess of relaxed circular DNA (rcDNA) intermediates. However, primer extension raising from the rcDNA in virus inoculates and intracellular nucleocapsids generates overlapping annealing products and results in identical products as cccDNA. This problem can be overcome by (I) prior enrichment of nuclei, (II) Hirt extraction to remove polymerase‐bound rcDNA, (III) digestion by plasmid‐safe DNase (PSD) showing some selective nuclease activity against rcDNA. However, nuclei isolation and PSD digestion are not completely selective against rcDNA. We found that cccDNA‐specific primers showed limited specificity when the ratio of rcDNA/cccDNA in samples from infected HepG2‐NTCP cells exceeded 100. Similarly, 400 copies of rcDNA purified from HBV stocks resulted in the same signal as one copy of supercoiled plasmid. Since current in vitro infections require high mge, early detection of cccDNA is affected. In infections of HepG2‐NTCP cells using high levels of virions, we observed that not only the rcDNA‐containing inoculum but also progeny intracellular rcDNA provided additional signals in a cccDNA quantification, when the samples were not previously treated with nucleases. We therefore tested a panel of nucleases and analysed their specificity to selectively hydrolyse rcDNA of virions without affecting circular plasmids. One of these nucleases, T5 exonuclease (T5 Exo) digested rcDNA faster and more efficiently than PSD but letting supercoiled DNA unaffected. Moreover, we combined T5 Exo treatment with our Taqman quantitative PCR and determined cccDNA and rcDNA kinetics in samples from in vitro infected HepG2‐NTCP, HepaRG‐NTCP and HepaRG cells. Taken together, our observations will be beneficial to laboratory detection and diagnostic analysis of cccDNA and helpful to study cccDNA early formation and accumulation in in vitro and in vivo infection systems. 211 Index of Authors A Aarnoutse, R. Abken, H. Abramowski, P. Abu Sin, M. Ackermann, C. Adamek, M. Addo, M. M. Adegnika, A. A. Adeoye, O. Adjabeng, M. Adu‐Sarkodie, Y. Aebischer, T. Ahmad, F. Ahmed, N. Ahrens, F. Aistleitner, K. Alabi, A. Alanjari, M. Alawi, M. Albarrak, A. M. Albarrak, M. M. Albig, H. Albrecht, J. Al‐Emran, H. Ali, S. Almasri, M. Altfeld, M. Ameres, S. Ammer, L. Anderl, F. Andraschko, M. Antes, I. Anton, G. Antwerpen, M. Apel, A. K. Arasteh, K. Arenz, L. Armean, S. Assiri, A. M. Atenchong, N. Athanasoulia, A. Atschekzei, F. Auer, R. Autenrieth, I. Azam, K. B Bachmann, M. Bachmann, O. Baines, J. Bakoua, D. Bange, F. Barac, P. Bartenschlager, R. Barth, T. Basic, M. 7 Bates, M. 109 Battefeld, W. 30 Bauer, S. 44 Bauer, A. 112, 146 Bauer, A. 199, 200 Bauer, T. 170 Baulig, A. 155 Baumann, K. 42 Baumert, P. 133 Baumgartner, T. 71 Bayer, P. 27 Becker, C. 53, 57 Becker, K. 145, 158 Becker, S. 1, 44, 53, 54 Beckert, P. 109 Becker‐Ziaja, B. 51 Beermann, S. 3 Beggel, B. 173 Behnke, M. 119, 131 Behrends, U. 98 Behrens, G. 187 Beissert, T. 44 Belmar Campos, C. 124 Benzler, J. 3 Berdien, B. 28 Berg, T. 175, 180 Berk, M. 16 Berkowski, C. 168 113 Berrocal Almanza, L. C. Beschorner, N. 29, 176 Bestehorn, M. 41, 46 Bhattacharjee, S. 68, 79 Bhuju, S. 200 Bialek, J. 195 Biedenkopf, N. 1 Biehl, L. M. 8, 124, 144 Bierbaum, G. 140, 144 Bila, C. 109 Bilitewski, U. 62, 67, 142, 147 Binger, T. 33, 105 Blatnik, R. 10 7 Bleich, A. Bleiziffer, S. 116 Blockus, S. 161 Blohm, U. 171 Bockholt, S. 60, 61 Boden, K. 30 Bodenstein, I. 140 Boeree, M. 14 Boesecke, C. 177, 190, 191, 202, 203 Bogner, J. 184, 190, 194 Bohm, K. 169 Bohne, F. 166, 178 Bonnet, M. 109 14 178 28 119 207 148 1 13 3 70 105 73 188, 192 113 110 35 109 148 90 33 33 76 89 130 109 33 186, 204, 205 88, 91 105 77 121 86 87 152 156, 157 165, 201 184 126 33 54 184 11 128, 135 22, 64, 147 109 75 72 7 104 23 149 32 48 212 Index of Authors Borregard, S. 1 Borrmann, S. 102 Böttinger, N. 178, 179 Bouhired, S. 151, 160 Bouyoukou Hounkpatin, A. L. 13 Brack‐Werner, R. 163, 185 Brandmüller, C. 91 Brauer, J. 3 Bremer, B. 172, 174 Bremer, C. M. 172 Brinkmann, F. 110 Brönstrup, M. 112, 146, 161 Brosnahan, J. 13 39 Brügge, B. Brüning, J. 81 Brunkhorst, F. M. 20 Bruns, T. 76 Bruske, E. 109 Buchholz, F. 29, 176 Budeus, B. 25 Budinger, S. 57 Buggisch, P. 175 Buhl, M. 72, 147 Büning, H. 196 Bunk, B. 128, 136 Burggraf, M. 49 Busch, D. 82, 87, 88, 89, 167 111 Busenbender, L. Butenop, J. 2 Buti, M. 25 C Carroll, M. 51 Cassens, J. 81 Castell, S. 37, 56 Chachage, M. 199, 200 Chakraborty, T. 19, 36, 100, 123, 127, 128 135, 136, 137, 143 Chanda, S. K. 49, 189 Chaudhry, Z. 92 Chemnitz, J. 29, 176 41, 42, 45, 46, 47 Chitimia‐Dobler, L. Christ, H. 8 Christner, M. 90, 144 Churchyard, G. 14 Cichutek, K. 44, 49, 189 Cicin‐Sain, L. 92 Ciesek, S. 54, 162, 180 Cirac, A. A. 98 Claus, H. 3, 119 Clowes, P. 200 Colbers, A. 14 Cooper, P. 50, 209 Corman, V. M. 33, 43 Cornberg, M. 172, 174, 175 Cornely, O. A. 8, 66, 85, 93, 94, 101 Cramer, J. P. 115 D Daglidu, E. 81 Dahlke, C. 1 Dähnert, L. 171 Dammermann, W. 176 Daniel, R. 50 Daschkin, C. 63 Däumer, M. 182 David, S. 162 Dawson, R. 14 De Gruyter, H. L. 4 de Mutsert, G. 4 Degen, O. 206 Deibert, J. 103 Dekker, D. 105, 130 Dembek, C. 170 Demirkaya, G. 78 Denecke, K. 3 Dennehy, K. 82 Depner, M. 95 Dersch, P. 62, 80 Des Portes, V. 189 Deterding, K. 174 Di Gennaro, M. 2 Diacon, A. 14 Diederich, S. 55 Dierks, P. 193, 207 Dietrich, U. 196 Dietz, J. 168 Dietzel, E. 54 Dimitriou, V. 85 Discher, T. 83 Dobler, G. 12, 34, 38, 40, 41, 42, 45, 46 Doijad, S. 36, 100 Domann, E. 136 Dornauer, S. 86 Dougan, G. 31 Draenert, R. 184, 190 Dreher, S. 88 Dressler, L. 10 Drexler, J. F. 43 Driesch, D. 201 Drosten, C. 33, 43 Dudakova, A. 120 Duddeck, A. 129 Dugan, H. 204 Dünzkofer, J. 97 Durán Graeff, L. A. 93 E 193, 206, 207 Eberhard, J. Eberhardt, K. 200 Eckmanns, T. 119 Edoa, J. R. 13 213 Index of Authors Eftime, D. Ehlers, C. Ehlkes, L. Eibach, D. Eick, J. Eickmann, M. Eiden, M. El‐Hadidi, M. Elmore, M. J. Enders, E. Engelhardt, M. Engels, I. Enkirch, T. Erfle, H. Esser, S. Etoka‐Beka, M. K. Evengard, B. Ewers, C. F Fabricius, A. Fackler, O. Faist, B. Falgenhauer, L. Falk, C. S. Farah, M. E. Farowski, F. Fast, C. Fätkenheuer, G. Feederle, R. Fehling, S. K. Fehse, B. Feig, M. Feihl, S. Feldt, T. Fesser, A. Festag, M. Feuchtinger, T. Fickenscher, H. Fiedler, A. Fink, L. Finke, S. Fischer, A. Fischer, D. Fischer, J. Fischer, N. Fischer, W. Fischl, W. Flach, B. Fobil, J. Fontaine, E. Formenty, P. Formichella, L. Foschi, F. Fouchier, R. F. Frahm, S. 68 Fraisse, L. 112 Frangoulidis, D. 116, 117 Frank, M. 109 Franke, A. 128 Frede, N. 95 Fritzenwanker, M. 135 Froidbise, A. 146 Fu, C. 146 Fux, R. 53 G Gajdiss, M. 140 Galaski, J. 192 Galle, P. 170 Gan, E. 153 Ganesan, R. 69, 81 204 Garcia‐Beltran, W. Garsevanidze, E. 37 Gärtner, K. 97 Garzetti, D. 124 Gastmeier, P. 21, 119, 122, 131, 133, 138 Gattenlöhner, S. 57, 83 Geldmacher, C. 199, 200 Gentil, K. 123, 137 Gerhard, M. 63, 72, 77, 79 Gerlach, R. 71 Germanaud, D. 189 Gertler, M. 2 Ghosh, H. 19, 100, 123, 128, 135, 137 Gieger, C. 87 Giehler, F. 86 Gil, H. 70 Gillespie, S. 14 Gillor, D. 190, 194 Git, A. 16 Gladstone, B. P. 118 100 Glaeser, S. Glaser, J. 71 Glässner, A. 191, 202, 203 Glebe, D. 172 Goeijenbier, M. 4 Goeijenbier, S. 4 Goeser, F. 65, 191, 202, 203 Goeser, T. 182 Goesmann, A. 128, 136, 143 Goffinet, C. 187 Gordts, S. 161 Gosten‐Heinrich, P. 73 Goyal, S. 113 Grallert, H. 158 170 Grambihler, A. Grassl, G. 7 Gräter, T. 48 Grein, F. 125, 134 Gressens, S. 204 150 111 70 70, 105, 130 78 1, 44 34, 171 22 55 3 8 134, 149, 154 153 32 182, 194 103 118 31 2 192 82 19, 36, 100, 123, 127, 128 135, 136, 137 172, 174 33 94 171 190, 194, 208 97 1 28 119 21, 116, 133 200 104 178, 179 82 128 44 57 162 66 34 69, 81 90 67 32 12, 38, 42 70 112 55 68 118, 126 4 214 Heitmann, L. Helfer, M. Hellman, J. Hellmich, M. Hemmrich‐Stanisak, G. Hengst, J. Henkel, L. Hentrich, M. Herold, S. Herrera‐Léon, S. Herrmann, E. Herrmann, J. Herzmann, C. Hesterkamp, T. Heyckendorf, J. Hiergeist, A. Higgins, P. G. Hilker, R. Hillenbrand, A. Hinkov, H. Hiscox, J. A. Hocke, M. Hoefler, D. Hoehne, B. Hoelscher, M. Hoffmann, C. Hoffmann, D. Hoffmann, D. Hoffmann, D. Hoffmann, S. Hofmeister, J. Hogan, B. Höland, A. Hölscher, M. Hölzl, F. Höner zu Siederdissen, C. Hoppe, S. Hörauf, A. Horn, J. Horn, S. Hos, N. Huber, A. Hubich‐Rau, S. Hübner, J. Hübscher, K. Hudecek, M. Hug, M. Hüppe, D. Huson, D. Huth, A. Hutzler, S. I Idelevich, E. Ihle, V. Ilori, E. Index of Authors Gresser, N. Grimbacher, B. Grimm, D. Grin, I. Gronbach, K. Groschup, M. H. Gross, H. Groß, H. Groß, U. Grosse, S. Grosset, J. Großmann, S. Grozdanovic, Z. Grundhoff, A. Grüner, B. Grützner, E. Gubbala, S. Guenther, S. Gunka, K. Günther, S. Günther, A. Günther, G. Günther, P. Günther, S. Gürtler, L. Gust, B. Gutiérrez, S. Gwozdzinski, K. H Haas, R. Haase, K. Häcker, G. Hadian, K. Hagel, S. Haid, S. Hain, T. Hamilton, S. Hammann, P. Hamprecht, A. Hardtke, S. Harmrolfs, K. Harms, H. Harmsen, D. Hartmann, P. Hasibuan, I. Hasreiter, J. Hauber, I. Hauber, J. Haverich, A. Heemann, U. Heide, L. Heikenwälder, M. Heinold, A. Heinrich, N. Heinz, F. X. 2 11, 95 27, 183, 185 6 64 34, 171 157 156 50, 120, 209 183 112 15 113 90, 176 48 184 204 60, 61 50 31, 51, 52, 55 59 106 82 31, 51, 52, 55 163 156, 157 69, 81 123, 127 67 200 132 86 66 161 36, 100, 136, 143 16 146 21, 133, 138 174, 181 23 149 128 81, 94, 108, 196 50 166 28, 29 28, 29, 176, 195, 196, 206 92 88, 89 156, 157 77, 183 25 12, 14, 38, 42 41 215 145 163 118 8 128 174 200 8 5, 44, 57 70 175 23, 112, 146 107 145 15, 110, 111 116 128 143 48 64 55 76 199 38 14, 38, 199, 200 194 25 85 200 102 111 70, 105 19 12, 42, 109 64 172 10 17, 65, 151, 160 26, 37 28 69 196 44 121, 129 95 178 132 175 22 8 44 145, 158 132 3 Kern, W. 21, 131, 132, 133, 138 Khallouf, H. 10 Kibiki, G. 14, 109 Kiehl, M. 8 Kienle, E. 185 Kim, L. 154 Kirchner, G. 3 Klann, F. 92 Kleine, C. 2 Klett‐Tammen, C. J. 56 Klevenz, A. 10 Klimka, A. 159 Kling, A. 112 Klinker, H. 175 Klintworth, A. 162 Klotz, C. 73 Klupp, E. M. 90 Knaack, D. 158 Knapp, B. 183 Knop, V. 175 Knops, E. 173, 182 Knüpfer, M. 12, 42 Koch, A. 72 Koch, J. 208 Koenig, R. 189 Koeppel, M. 71, 124 Kofla, G. 8 Köhler, P. 93, 101 Kokordelis, P. 191, 202, 203 Kombo, M. 103, 104 König, G. M. 149, 151, 160 König, R. 49 Kopp, K. 86 Koppensteiner, H. 163 24, 25 Kosinska, A. Koukouikila‐Koussounda, F. 103, 104 Koup, R. 199 Kowuor, D. 199 Kraft, A. R. 172 Krähling, V. 1 Krämer, B. 65, 191, 202, 203 Kratzer, W. 48 Krause, G. 3, 37, 56, 169 Kräusslich, H.‐G. X Kreijtz, J. 4 Kreitmeyr, K. 121 Kremmer, E. 97 13, 102 Kremsner, P. Kreuels, B. 70 Kreuz, D. 44 Krismer, B. 141, 145 Kröger, N. 90 Kroidl, A. 199 Krönke, M. 69, 159 Krumkamp, R. 105, 130 Krut, O. 159 Index of Authors Imirzalioglu, C. 19, 36, 100, 123, 124, 127 128, 135, 136, 137 Indenbirken, D. 90, 176 Ingiliz, P. 177, 191 Isherwood, L. 109 J Jacobs, R. 188 Jaeger, A. 105 Jäger, C. 24 Jakob, M. 20 Jambrecina, A. 1 Jankauskaite, L. 5 Janke, M. 3 Janssen, I. 50 53 Jany, S. Jazmati, N. 72, 132 Jean Claude, D. A. 13 Jeffreys, L. 165 Jensen, B. 194 Johnsen, J. 7 Jorge, A. 142 Josephs, S. 200 Jost, S. 204 Josten, M. 140 K Kaczmarek, D. 65, 202, 203 Kaderali, L. 32 Kaersten, S. 119 Kahlhofer, C. 116, 117 173, 182 Kaiser, R. Kalaghatgi, P. 173, 182 Kalinke, U. 44 Kallis, S. 32 Kalsdorf, B. 15, 110, 111 Kammel, C. 185 Kämpfer, P. 100 Karch, A. 20, 37, 59, 129 Karch, H. 80 Karimova, M. 176 Karimzadeh, H. 25 Karpinski, J. 29 Karthaus, M. 8 Kasmapour Seighalani, B. 92 Kasonta, R. 1 Kastenbauer, U. 74 Katchanov, J. 165, 201 Kayser, S. 82 Kaysser, L. 155, 157 Keiser, J. 68 Keitel, V. 182 Keller, L. 146 Kellner, M. 97 Kerber, R. 55 216 Machowicz, R. 28 Magassouba, N. 51 Malainou, C. 83 Mall, S. 3 Malo, A. 167, 178, 179 Mangu, C. 12, 38 Mankertz, A. 190 Manns, M. 54, 72, 162, 172, 174, 180 Mapamba, D. 109 Marahiel, M. A. 127 Marks, F. 130 Martin, L. 96 Martrus, G. 205 Mast, Y. 157 Matevossian, E. 89 Matthews, D. A. 55 Matuschek, A. 2 Maurer‐Stroh, S. 153 Mauss, S. 175, 177 Mautner, J. 98 May, J. 70, 105, 130 57 Mayer, K. Mcharo, R. 199 Meichsner, J. 27 Mejías‐Luque, R. 77 Melkova, Z. 49 Mellet, K. 14 Memish, Z. A. 33 Mertins, S. 159 Meyer, B. 33 Meyer, H. 63 Mfinanga, S. G. 39 Michler, T. 24, 183 Mikolajczyk, R. T. 20, 26, 37, 59, 129 Milic, J. 78 14 Minja, L. T. Mischnik, A. 21, 132, 133 Mnkai, J. 199 Mock, U. 28 Mockenhaupt, S. 183 Moesker, F. M. 4 Momburg, F. 166 Mondesert, G. 146 Monjarás Feria, J. 75 Moog, U. 30 Moosmann, A. 91, 96 Morales‐Nebreda, L. 57 Mordmüller, B. 13, 102 Morty, R. 57 88 Mossmann, A. Mouwenda, Y. D. 13 Moyer, D. 3 Mraheil, M. A. 127 Mshana, S. E. 50 Msila, H. 12, 38 Mühlebach, M. D. 44 Index of Authors Kuehn, A. Kuffour, E. O. Kulik, A. Kummer, S. Kümmerle, T. Kupke, A. Kurth, A. Kurz, M. Kurzawa, N. Kusumawati, R. L. L Laedtke, T. Lagrange, S. Laketa, V. Lala, B. Lamshöft, M. Lang, V. Lange, C. Lange, U. Langebartels, G. Langhans, B. Lattwein, E. Lecuona, E. Lehmann, C. Lehmann, M. Leipoldt, F. Lell, B. Lellek, H. Lempp, F. A. Lengauer, T. Lerch, M. Levin, M. Liekweg, A. Lin, H. Ling, L. L. Liss, B. Loew‐Gil, E. Loffredo‐Verde, E. Lohmeyer, J. Lohse, A. W. Loik, S. Lucic, B. Lüdtke, A. Lugert, R. Lukat, P. Luo, S. Lusic, M. Lütgehetmann, M. Lüth, S. Lutz, J. Lutz, P. Lutz, T. M Maboko, L. 87 200 141 207 190 44, 53 55 146 27 50 3 112 27 113 3 50 15, 106, 107, 110, 111 195 132 65 33 57 190, 208 4, 91 155 13, 109 90 210, 211 173, 182 66 16 132 187 154 85, 93 77 68, 167 5, 57, 83 1, 66 2 197, 198 51, 52, 55, 60, 61 209 112 68 197, 198 90 176 89 65, 191, 202 177 12, 38, 42, 199, 200 217 Okamo, B. 50 Olaru, I. D. 15, 110, 111 Omrani, A. S. 33 Opare, D. 70 Ortlepp, J. R. 50 Osterhaus, A. D. 4 Overmann, J. 36, 50, 100, 128, 136 Owusu‐Dabo, E. 105 120 Öztürk Mert, S. P Pallasch, E. 60, 61 Panayotova‐Dimitrova, D. 187 Panse, J. 8 Parcina, M. 65 Passmann, S. 168 Paszkiewicz, P. 179 Pati, N. B. 36, 100 Paul, J. 2 Pavle, K. 3 Pawlita, M. 199 Pazia Juma, S. 109 Pecar, A. 121 Pena Diaz, L. A. 119 Peper, J. 82 Perchermeier, S. 114 Perner, D. 168 Perscheid, C. 3 Peschel, A. 139, 141, 142, 145 Peter, S. 21, 22, 64, 133 Peters, G. 158 Petersen, J. 175 Peuser, I. 182 Pfarr, K. 17, 151, 160 Pfister, H. 209 Phillips, P. 14 Phiri, I. K. 39 31 Pickard, D. J. Pietschmann, T. 161 Pleschka, S. 57, 164 Pletz, M. W. 30 Plum, G. 81, 108 Podola, L. 200 Poggensee, G. 3 Pohlentz, G. 80 Pollakis, G. 200 Poso, A. 6 Poulain, P. 103 Prazeres da Costa, C. 17, 39, 68, 79, 167 Prior, K. 128 Protzer, U. 24, 25, 166, 167, 170 178, 179, 183 Prüfer, S. 44 Index of Authors Mühlen, S. Müller, R. Müller, A. Müller, C. Müller, F. Müller, K. Müller, M. A. Mundt, S. Muñoz‐Fontela, C. Muth, D. Müthing, J. Mutlu, G. Mutter, W. Muzillo, D. Mwakatima, M. Mwansongela, C. Mwape, K. E. Mwinuka, N. N Nadai, Y. Nadeu Prat, F. Nattermann, J. Neha, M. Nelson, C. Nelson, M. Neu, A. S. Neubauer, H. Neuenhahn, M. Neumann‐Fraune, M. Newton, S. M. Ngowi, B. Ngowi, H. Ni, Y. Nickel, F. Nickl, M. Nicolai, A. Niedermeyer, T. Niehrs, A. Niemann, S. Nischalke, H. D. Nitiu, R. Nolden, T. Nolting, A. Normahoomed, E. Nouatin, O. P. Nowag, A. Ntinginya, N. E. Ntoumi, F. Nunn, A. Nusser, D. O Obermann, B. Oestereich, L. Ojo, O. 62 23, 112, 146 134, 154 164 93 34 33 23 51, 52, 60, 61 33, 55 80 57 145 181 199 12, 38 39 199 200 125 65, 191, 202, 203 188 3 177 108 30 82, 88, 89 182 16 39 39 211 159 27 159 23, 157 205 109 65 63 162 1 39 13 108, 196 14, 42, 109 103, 104 14 125 21, 133 51, 52, 60, 61 3 218 Sanchez‐Carballo, P. Sanchez‐Padilla, E. Sanne, I. Sarfo, F. S. Sarpong, N. Sarrazin, C. Sauber, S. Schäberle, T. F. Schäfer, C. Schäfer‐Eckart, K. Schapranow, M. Schaprian, A. Schattenberg, J. Schaufler, K. Scheikl, D. Scherübl, H. Scherzinger, A. Schessner, J. P. Scheuplein, V. A. Scheurich, C. Schiefer, A. Schiell, M. Schiemann, M. Schimanowski‐Thomson, H. Schindele, F. Schindler, D. Schindler, M. Schirmacher, P. Schlaphoff, V. Schleenvoigt, B. T. Schleicher, A. Schleimer, N. Schlosser, J. Schlott, F. Schmidberger, J. Schmidt, F. Schmidt, J. Schmidt, R. E. Schmidt, V. Schmiedel, J. Schmiedel, S. Schmitz, H. Schmitz, R. P. Schmoldt, C. Schmoock, G. Schneefeld, M. Schneider, M. Schneider, T. Schneiderhan, W. Schnödt, M. Schober, E. Schols, D. Scholten, S. Scholz, H. Schommers, P. Index of Authors Q Qu, B. Quitt, O. Qurishi, N. R Rambaut, A. Raupach‐Rosin, H. Rausch, M. Rausch, M. Rausch, P. Rebensburg, S. Rehal, S. Reiberger, T. Reischl, U. Reither, K. Renders, L. Reuken, P. Rezazadeh, Y. Rieble, L. Rieger, T. Riehm, J. Riemer, A. B. Riess, M. Riess, M. Rimmelzwaan, G. F. Rinder, M. Ring, W. Rinker, F. Rißner, F. Rizzetto, M. Robinson, N. Rockstroh, J. Röder, N. Roggendorf, M. Rohde, A. M. Rohde, H. Rohrmann, K. Rojas, J. Rolling, T. Rossmann, F. Roth, M. Rübsamen, N. Ruggieri, A. Ruibal, P. Rupnik, M. Rupp, J. Russo, C. Rwegoshora, F. Rybniker, J. S Saathoff, E. Sahin, U. Salzberger, W. Sammet, S. 211 166 182 55 37, 129 134 175, 177 7 163 14 177 116, 117 14 89 76 136 210 60, 61 58 10 49 49 4 34 78 172 20 25 69, 81 177, 190, 191, 202, 203 24, 183 25 21, 122, 131, 133, 138 90, 124, 144 162 95 115 99 2 37 32 51, 52, 61 50 72 170 199 108 200 44 204 190 219 111 109 14, 109 200 105 168, 175, 180 153 154 29, 195, 196 8 3 143 170 31 63 78 145, 158 10 44 207 151, 160 146 200 12 67 84, 87 163 87 174 30 194 158 171 88, 89 48 27, 185 44, 53 11, 188, 192 39 19, 123, 137 1, 115 189 20 5, 57 30 23 163, 185 134, 149, 154 50 196 91 161 177, 182 58 186, 194 Stanic, M. Stanke, F. Stankov, M. Stecher, B. Steck, N. Stegmann, E. Steib‐Bauert, M. Steinbach, A. Steinberg, J. Steinhagen, K. Steinke, K. Stepek, J. Steubl, D. Stevanović, S. Stiasny, K. Stich, A. Stirner, R. Stocker, H. Stoecker, K. Strassburg, C. P. Straubinger, K. Strecker, T. Stripecke, R. Stubbe, H. C. Stückler, F. Stürz, I. Suerbaum, S. Suhr, C. Sumanlatha, G. Susser, S. Sutter, G. Suwandi, A. Süzer, Y. Sznajder, J. T Tacconelli, E. Tacke, D. Terhalle, E. Theisen, M. Thoeringer, C. Thoma, B. Thomas‐Morr, P. Tibroni, N. Timm, J. Ting, J. P. Titz, A. Tolosa, E. Tom‐Aba, D. Tominski, D. Tondera, C. Torres, L. Tóth, I. Toti, L. Trautner, J. Tufa, D. M. Index of Authors Schorpp, K. 86 Schröder, C. 11 Schröder, W. 118, 126 Schrödl, K. 184 Schroeder, N. 199 Schulte, B. 72 Schulz, T. 161 Schulze zur Wiesch, J. 176, 206, 207 Schulze, M. H. 120, 209 Schumann, R. R. 113 Schummer, D. 146 Schuster, D. 140 Schütz, M. 22, 147 21, 138 Schwab, F. Schwarz, N. 3 Schwarze‐Zander, C. 190, 191, 202, 203 Schweers, J. M. 147 Schweickert, B. 119 Schwengers, O. 136, 143 Schwertner, C. 78 Seeger, W. 57 Seidel, J. 7 Seifert, H. 21, 128, 131, 132, 133, 135, 138 Seifried, J. 44, 49, 189 Selvakumar, B. 57 Semmler, T. 31 Serr, A. 133 Seybold, U. 190 Shams‐Eldin, H. 53 Sheikh Ali, H. 171 Sib, E. 140 Siddiqui, N. 16 33 Sieberg, A. Siebert, A. 125 Sieper, T. 35 Sierra, S. 173, 182 Sierra‐Aragón, S. 173 Sievers, C. 169 Sikasunge, C. S. 39 Silling, S. 209 Simoneau, C. 204 Sina, C. 72 Slevogt, H. 113, 165, 201 Smitsman, N. 15, 111 Solbach, P. 72 Sommer, H. 118 Song, F. 53 116 Specht, K. Spengler, U. 65, 191, 202, 203 Spinner, C. 177, 194 Spohn, K. 126 Sprague, L. 30 Sprinzl, M. 170, 175 Spröer, C. 128 Stahl, F. R. 1 Stallmach, A. 66, 76 220 198 180 187 71, 124 7 150, 157 132 10 78 33 148 69 88, 89 82 41 2 184 165, 201 35 65, 190, 202 114 1, 55 97 1 183 35 72 187 113 168, 175 4, 53, 91 7 44 57 72, 118, 126 66, 101 15, 107, 110 13 72 116 144 192 173 49 18 206 3 165, 201 44, 189 199 206 146 55 188 Wiegmann, B. 92 Wieland, U. 190 Wieler, L. H. 31 Wiese‐Posselt, M. 122, 131, 138 Wiesner, C. 23 Will, U. 76 Wille, T. 21 Willmann, M. 22, 64 Wink, J. 146 Winkler, A. S. 39 Winter, G. 145 Winter, J. 10 Winter, S. 108 Wisplinghoff, H. 85, 93, 101 Wisskirchen, K. 178, 179 Witte, T. 11 Wohlleben, W. 150, 157 Wolf, F. 155 Wolf, T. 57, 194 Wölfel, R. 35, 55 Wolff, T. 57 Wolke, M. 81 Wolter, F. 202, 203 Wright, J. 16 Wühl, M. 10 Wurr, S. 60, 61 Wyen, C. 194 Y Yao, Y. 36, 100, 128, 135, 136, 137, 143 13 Yazdanbakhsh, M. Yennan, S. 3 Yoh, H. 49 Yoh, S. M. 49, 189 Z Zaburannyi, N. 112 Zettler, J. 155 Zeuzem, S. 168, 175 Ziebuhr, J. 164 Ziegler, U. 34 Zielinski, C. 9 Ziemert, N. 148, 157 Zimmermann, O. 50 Zimmermann, T. 170 Zinser, M. 1 Zoch, B. 59 77 Zöller, J. Index of Authors U Ulrich, R. G. Urban, S. V Vadasz, I. Valentini, C. Valluri, V. van Bömmel, F. van de Vijver, D. A. van den Dries, L. van Gumpel, E. van Lunzen, J. Vehreschild, J. J. Vehreschild, M. J. G. Vergara‐Alert, J. Vermehren, A. Vermehren, J. Verschoor, A. Viegas, S. Vina‐Rodriguez, A. Vinnemeier, C. D. Voges, M. Voigt, E. Völker, F. Vollmar, P. Volz, A. von Bernuth, H. von Both, U. von Buttlar, H. von Eiff, C. von Hahn, T. von Messling, V. von Müller, L. Vouvoungui, J. C. W Wagner, S. Wagner, T. Waibler, Z. Walker, A. Walter, H. Walter, M. Wasmuth, J.‐C. Wassilew, N. Weber, T. Wedemeyer, H. Weidenmaier, C. Weikum, J. Weinmann, A. Weis, M. eismüller, T. Welzel, T. Wendtner, C. Wenzel, S. Westhaus, S. 171 210, 211 57 197, 198 113 175 4 4 108 29, 206 93, 101, 132, 190 8, 66, 72, 93, 101, 124 44 175 175 68 109 171 115 195 191 209 116 4, 53 110 16, 121 152 158 54, 162, 180, 182 153 50 103, 104 6, 75 17 44 173 173 116, 117 194 106, 107, 110, 111 150 172, 174, 175, 181 22, 145 125 170 152 202 175 8 112 162, 180 221 Speziell für die Therapie
der Clostridium-difficileassoziierten Diarrhoe
entwickelt
➔ 97 Prozent Therapieerfolg und
geringe Rückfallquote *
➔ gleichmäßige Freisetzung des Wirkstoffs
dank monolithischem Kapselkern
➔ gute Verträglichkeit
durch geringes Resorptionsrisiko **
RIEMSER Pharma GmbH | An der Wiek 7 | 17493 Greifswald – Insel Riems
phone +49 30 338427-0 | fax +49 30 89748045 | [email protected]
www.VANCOMYCIN.de
* Vancomycin ENTEROCAPS ® vs. Metronidazol: 97 % vs. 76 % der Patienten mit schwerer CDAD waren geheilt; p = 0,02 (Zar et al., Clin Infect Dis 2007; 45: 302–7).
** Bitte berücksichtigen Sie die Fachinformation (Stand: August 2015).
PFLICHTANGABEN gem. § 4 HWG – Vancomycin ENTEROCAPS® 250 mg – Wirkstoff: Vancomycinhydrochlorid – Zusammensetzung: 1 Hartkapsel enthält 250 mg Vancomycinhydrochlorid (entsprechend mindestens 262.500 I.E. Vancomycin).
Sonst. Bestandt.: Macrogol 6000, Gelatine, Titandioxid (E 171), Indigocarmin (E 132), Eisenoxid (E 172), Schellack, Propylenglykol, Kaliumhydroxid, konzentrierte Ammoniaklösung. – Anwendungsgebiete: Vancomycin ENTEROCAPS® 250 mg sind zur
Behandlung von Enterokolitiden hervorgerufen durch Clostridium difficile (Clostridium difficile assoziierte Diarrhoe und Enterokolitis), Staphylokokken (Staphylokokken-Enterokolitis) geeignet. Bei anderen Infektionen ist Vancomycin, wenn es oral
angewendet wird, nicht wirksam, da es aus dem Magen-Darm-Trakt nicht nennenswert resorbiert wird. – Gegenanzeigen: Wenn Sie allergisch gegen Vancomycin oder einen der sonstigen Bestandteile dieses Arzneimittels sind. – Nebenwirkungen:
Erkrankungen des Gastrointestinaltrakts: Selten kann Übelkeit auftreten. – Da Vancomycin nach Einnahme im Allgemeinen nicht in wirksamen Mengen aus dem Magen-Darm-Trakt in das Blut übergeht, sind Nebenwirkungen, wie sie nach intravenöser
Anwendung berichtet wurden, nach Einnahme der Hartkapseln im Allgemeinen nicht zu erwarten. Es besteht jedoch die Möglichkeit, dass gelegentlich bei Patienten mit Entzündung der Darmschleimhaut nach wiederholter Einnahme wirksame
Vancomycin-Konzentrationen im Blut auftreten, vor allem wenn gleichzeitig die Nierenfunktion eingeschränkt ist. – Bei längerer Einnahme von Vancomycin kann es zu einem vermehrten Wachstum von Krankheitserregern kommen, gegen die Vancomycin nicht wirksam ist. Daher sollte der behandelnde Arzt auf Anzeichen einer erneuten Infektion achten. – Nach intravenöser Gabe von Vancomycin wurden folgende Nebenwirkungen berichtet: Infektionen: Häufig wurden orale Candidosen
beobachtet. Erkrankungen des Blutes und des Lymphsystems: Verminderung der Zahl bestimmter weißer Blutkörperchen (Neutropenie und Einzelfälle von Leukopenien) oder der Blutplättchen (Thrombozytopenie), Anstieg bestimmter weißer Blutkörperchen (Eosinophilie). Leber- und Gallenerkrankungen: In Einzelfällen sind erhöhte Leberenzyme, Hepatitis und Ikterus aufgetreten. Erkrankungen des Nervensystems: Schwindel kann auftreten. Es wurden Einzelfälle von Taubheitsgefühl (Parästhesien), Schläfrigkeit (Somnolenz), Krämpfe (Konvulsionen), Kopfschmerzen und Zittern (Tremor) beobachtet. Erkrankungen der Niere und Harnwege: Nierenversagen, hauptsächlich erkennbar an erhöhten Serumkreatinin- oder Blutharnstoffstickstoffkonzentrationen, Nierenentzündung (interstitielle Nephritis). Erkrankungen des Ohres und des Labyrinths: Hörverlust, Ohrenklingen. Überempfindlichkeitsreaktionen: schwere Überempfindlichkeitsreaktionen mit Kreislaufbeteiligung, Hautausschlag,
einschl. schwerer Formen von Hautentzündung (exfoliative Dermatitis, lineare (bullöse) IgA Dermatose, Stevens-Johnson-Syndrom), Arzneimittelfieber, Schüttelfrost und Eosinophilie (Anstieg bestimmter weißer Blutkörperchen), in Einzelfällen Gefäßentzündung. Während oder kurz nach rascher intravenöser Infusion von Vancomycin können Überempfindlichkeitsreaktionen einschl. Blutdruckabfall, Atemnot, Nesselfieber oder Juckreiz auftreten. Es kann auch zu Hautrötung am Oberkörper („red neck“)
oder Schmerzen und Krämpfen der Brust- und Rückenmuskulatur kommen. In einem Fall wurde eine solche Reaktion auch nach der Einnahme von Vancomycin berichtet. – Warnhinweise: Arzneimittel für Kinder unzugänglich aufbewahren. –
Verschreibungspflichtig – Stand der Information: August 2015 – Pharmazeutischer Unternehmer: RIEMSER Pharma GmbH, An der Wiek 7, 17493 Greifswald-Insel Riems, Deutschland
Notes XVII Social Programme Social Evening (Boarischer Abend) Date: 19 November, 2015 Time: 19:30 Location: Paulaner am Nockherberg Raum Bayern Hochstrasse 77 • 81541 München Fee: 50 EUR/Regular; 30 EUR/Student Guided night tour through Munich Date: 20 November, 2015 Time: 20:00 Meeting Point: Marienplatz, at the Mariensäule From the venue take either Bus 52 from station “Mariahilfplatz” direction “Marienplatz” or Tram 37 from station “Ostfriedhof” direction “Petuelring” and change at “Sendlinger Tor” to subway U3 direction “Olympiazentrum” and leave at “Marienplatz”. Fee: included General Information Certification The Joint Annual Meeting of the German Society of Infectious Diseases (DGI) and German Center for Infection Research (DZIF) is accredited by the Medical Chamber of Bavaria with 18 CME points of Category B. Additionally it is registered for 20 iCME points at the Academy of Infection Medicine. Poster Sessions All posters have to be mounted on Thursday, 19 November, 2015 until 13:00 and should be removed by Friday, 20 November, 2015 until 19:00. Location of Rooms from venue Lecture Hall Saal 1: Ground Floor Kastanienstube: First Floor Foyer 1 and 2: Ground Floor Kachelofenstube: First Floor First Floor Saal 2: Ground Floor Raum Bayern: Travel to Paulaner am Nockherberg By Public Transport From Munich Airport Take Tram S8 direction “Hersching” and dismount at station “München Rosenheimer Platz”. From there it takes 15 min. by foot. From Central Station Take either subway U1 direction “Mangfallplatz” or subway U2 direction “Messestadt Ost” and leave at “Kolumbusplatz”. From there it takes 10 min. by foot. Further information can be found at www.mvv‐muenchen.de. By Car Address for navigation system: Hochstrasse 77 81541 Munich WIFI Access Free WIFI is available throughout the whole congress area. SSID: Joint_Meeting Password: dgidzif XVIII