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GENETIC RELATIONSHIPS OF BACILLUS ANTHRACIS STRAINS ASSOCIATED WITH INJECTIONAL ANTHRAX IN

Robert Koch-Institut Centre for Biological Security 2. GENETIC RELATIONSHIPS OF BACILLUS ANTHRACIS STRAINS ASSOCIATED WITH INJECTIONAL ANTHRAX IN DRUG CONSUMERS. Roland Grunow, PhD, MD et al. RKI, Head of the Division Highly Pathogenic Microorganisms Director and Professor

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GENETIC RELATIONSHIPS OF BACILLUS ANTHRACIS STRAINS ASSOCIATED WITH INJECTIONAL ANTHRAX IN

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  1. Robert Koch-Institut Centre for Biological Security 2 GENETIC RELATIONSHIPS OF BACILLUS ANTHRACIS STRAINS ASSOCIATED WITH INJECTIONAL ANTHRAX IN DRUG CONSUMERS Roland Grunow, PhD, MD et al.RKI, Head of the Division Highly Pathogenic Microorganisms Director and Professor E-mail: grunowr@rki.de Workshop on the Biology of Anthrax National Museum of Wales, Cardiff, Wales, UK Date:  11th and 12th March 2014

  2. History of Anthrax in Heroin Consumers Differential diagnosis of infections with Staphylococcus aureus, Streptococus pygenes and Clostridium species Palmateer NE et al. Infections with spore-forming bacteria in persons who inject drugs, 2000-2009. Emerg Infect Dis. 2013 Jan;19(1):29-34. – 295 infections (157 C. botulinum, 33 C. tetani, 92 C. novyi, 13 B. anthracis) 2000 NorwayRingertz SH et al. Injectional anthrax in a heroin skin-popper. Lancet. 2000;356(9241):1574-5 A gram-positive endospore-forming aerobic rod was isolated from the soft tissue and cerebrospinal fluid; confirmation of Bacillus anthracis was made by PCR. “Since contaminated heroin was the probable source of infection, this case is of concern and warrants surveillance.” 49-year-old HIV-negative heroin skin-popper who lived in urban Oslo, Norway, single deadly infection with a clinical history of 12 days

  3. Case Definition of Injectional Anthrax Clinical confirmation (one of two criteria): • Signs of inflammation/infection (erythema, edema, abscess) in the region of drug injection • Necrotic fasciitis. Laboratory confirmation • Direct by isolation of B. anthracisor detection by PCR • [Indirect by detection of specific antibodies against B. anthracisand increasing titer] Epidemiological confirmation • Usage of substances or instruments with laboratory confirmation of a contamination with B. anthracis[suspecion]

  4. Anthrax Cases Since 2000in Heroin Consumers ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? Retrospective analyses of literature: One patient already in 2000! Ref.: Grunow R et al. , Euro Surveill. 2013 Mar 28;18(13).

  5. Seven Detected Anthrax Cases in Germany 2009 - 2012 • Dec. 2009, Aachen • March 2010, Aachen • March 2010, Passau • June 2012, Regensburg • June 2012, Regensburg • June 2012, Berlin • September 2012, Berlin 6,7 1,2 4,5 3

  6. Confirmation of Anthrax in IDU in Germany Reference of photographs: Grunow et al., Deutsches Aerzteblatt, 2012,49 Case 5 Intra-operative situationafter dissection of necrosis Case 6 Ulcus after repeated operative debridement Case 5 Multiple intra- and paravasale injection sides

  7. Objectives of Our Study • Serological pilot study to discover probably not detected anthrax cases in heroin users • Genotyping of isolates from 2000 – 2012 to make a comparative analysis • Conclusions on the duration and probable intensity of the outbreak event

  8. Serology Pilot Study • Two serum collections: • Pilot study by RKI „Drugs and chronic infections” (DRUCK), survey behavior and infection with HIV, Hepatitis B and C – response driven sampling approach; mainly in Berlincapillary blood samples dripped on special filter paper (Dried Blood Spots, DBS), n=244 • Sampling during initial health check heroin-consuming prisoners in 20 correctional facilities located in Baden-Wuerttemberg (in cooperation with Ministry of Justice); intravenous blood serum, n=44 • Questionnaire • Laboratory investigation of sera: accredited in-house anti-PA-ELISA (specificity 100%; sensitivity 1:16000 for sera, 1:24000 for plasma)accredited in-house anti-PA-Western blot (specificity 100%; sensitivity 1:200000 for sera, 1:100000 for plasma)

  9. Serology Study – Clinical Symptoms Table: Soft tissue symptoms among DRUCK participants (n=244) and probands from correctional facilities (n=44), RKI study in Germany 2010–2011 n. a. not applicable

  10. Serology Study Table: Summary of serological results, serum collection DRUCK and serum collection correctional facilities, Germany 2010–2011 No difference between both serum collections Right places; right persons?

  11. Anthrax Strains for Molecular Investigations a A112a and A112b refer to different colony morphologies of the same isolate b only DNA from clinical sample (skin swab) available ctwo isolates from the same case; Ringertz SH et al. Injectional anthrax in a heroin skin-popper, Norway, 2000. Lancet. 2000;356(9241):1574-5. Epub 2000/11/15.

  12. Molecular Identification

  13. Molecular Typing MLVA 8 Fragment lengths of MLVA-8 markers present in isolates from different years are shown according to Keim et al. (2000) MLVA-8 is suitable for cluster analyses

  14. Molecular Typing MLVA 31 High resolution MLVA-31 is suitable for strain analyses

  15. Strain Year of Country Bams 30 - pXO2 - AT - designation isolation repeats repeats A315 2000 Norway 75 9 Ba4599 2009 Scotland 75 7 A112a 1 2009 Germany 72 7 A112b 1 2009 Germany 75 7 A138 2010 Germany 75 7 UR - 1 2012 Germany 75 8 UR - 2 2012 Germany 75 8 A294 2012 Denmark 75 8 A306 2012 Denmark 75 8 A303 2 2012 Germany 75 8 MLVA Results on Bams30 and pXO2-AT 1 A112a and A112b refer to different colony morphologies of the same isolate 2 only DNA from clinical sample (skin swab) available

  16. Molecular Typing 2000-2012 • MLVA-8: all isolates belong to Cluster A1.b (Keim et al., 2000) • only difference: pXO2 • MLVA-31: all isolates belong to Cluster A, NEW GENOTYPE • only difference: pXO2 and Bams30 • not performed for „Norway“ strain from 1983

  17. Clustering of Isolates Dendrogram based on MLVA-31 typing of 904 isolates from Europe, Asia and Southern Africa. The „heroin anthrax“ isolates belong to the European A cluster. Ref.: Grunow R et al. , Euro Surveill. 2013 Mar 28;18(13).

  18. SNP-Analyse Ref.: Grunow R et al. , Euro Surveill. 2013 Mar 28;18(13). 27. Van Ert MN, Easterday WR, Huynh LY, Okinaka RT, Hugh-Jones ME, Ravel J, et al. Global genetic population structure of Bacillus anthracis. PLoS One. 2007;2(5):e461. 28. Price EP, Seymour ML, Sarovich DS, Latham J, Wolken SR, Mason J, et al. Molecular Epidemiologic Investigation of an Anthrax Outbreak among Heroin Users, Europe. Emerg Infect Dis. 2012;18(8):1307-13.

  19. Full Genome Sequencing of Anthrax Strains 1Rückert C et al. Draft genome sequence of Bacillus anthracis UR-1, isolated from a German heroin user. J Bacteriol. 2012 Nov;194(21):5997-8.

  20. Conclusions (1) • Several pathogens could cause anthrax-like clinical manifestations; therefore appropriate laboratory confirmation is required. • Laboratory diagnosis could be conducted by molecular detection using real-time PCR and confirmation by isolation of the pathogen with subsequent identification of bacteria by classical microbiology, PCR and MALDI-TOF. • Serological methods like ELISA and Western blot are important tools for epidemiological investigation and supporting elements for confirmation of diagnoses. • A pilot study with a limited sample number and geographical distribution did not reveal probable additional cases in Germany, but more extensive research could be helpful.

  21. Conclusions (2) Molecular methods for genotyping (MLVA 8/31, SNP-analyses) identified all studied isolates as the same strain, including the Norwegian isolate from 2000. It can be concluded that most likely the outbreak has been going on since at least 2000 with a probable similar source of contamination which might be still active. It is not clear how many cases have remained undetected since this time. So far, awareness by physicians and patients is most important for an early and effective treatment.

  22. Acknowledgement From Roland Grunow1 to all co-authors of the presentation • Gregor Grass2, • Wolfgang Beyer3 • David M. Wagner4 • Talima Pearson4 • Udo Reischl5, • Per Sandven6 • Anne Kjerulf7 • Mandy Elschner8 • Matthias Hanczaruk2 • Paul Keim4 • Silke R. Klee1 1Robert Koch-Institute (RKI), Centre for Biological Threats and Special Pathogens (ZBS2), Berlin, Germany; 2Bundeswehr Institute of Microbiology, Munich, Germany; 3Institute of Environmental and Animal Hygiene, University of Hohenheim, Stuttgart, Germany; 4Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, U.S.A.; 5Institute of Clinical Microbiology and Hygiene, University Hospital Regensburg, Regensburg, Germany; 6Norwegian Institute of Public Health, Department of Bacteriology and Immunology, Oslo, Norway; 7Statens Serum Institut, National Institute for Health Data and Disease Control, Copenhagen, Denmark; 8Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Jena, Germany Thank you for your attention

  23. Clusteranlyse anhand der MLVA-31-Profile The F-complex comprises the eight drug-related isolates.

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