GENETIC RELATIONSHIPS OF BACILLUS ANTHRACIS STRAINS ASSOCIATED WITH

INJECTIONAL ANTHRAX IN DRUG CONSUMERS

Robert Koch-InstitutCentre for Biological Security 2

Workshop on the Biology of Anthrax

National Museum of Wales, Cardiff, Wales, UK

Date:  11th and 12th March 2014

Roland Grunow, PhD, MD et al.RKI, Head of the Division Highly Pathogenic Microorganisms

Director and ProfessorE-mail: grunowr@rki.de

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 whoinject 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

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. anthracis or detection by PCR• [Indirect by detection of specific antibodies against B. anthracis and increasing titer]

Epidemiological confirmation•Usage of substances or instruments with laboratory confirmation of a contamination with B. anthracis [suspecion]

Ref.: Grunow R et al. , Euro Surveill. 2013 Mar 28;18(13).

Anthrax Cases Since 2000in Heroin Consumers

Retrospective analyses of literature:One patient already in 2000!

? ? ? ? ? ? ? ? ? ? ? ? ? ? ?

Seven Detected Anthrax Cases in Germany

1. Dec. 2009, Aachen2. March 2010, Aachen3. March 2010, Passau

4. June 2012, Regensburg5. June 2012, Regensburg6. June 2012, Berlin7. September 2012, Berlin

3

6,7

1,2

4,5

2009 - 2012

Confirmation of Anthrax in IDU in Germany

Case 5Multiple intra- and paravasale injection sides

Case 5Intra-operative situationafter dissection of necrosis

Case 6Ulcus after repeated operative debridement

Reference of photographs: Grunow et al., Deutsches Aerzteblatt, 2012,49

No Case Isolation PCR serology status

1 Dec. 2009, Aachen + + ND died

2 March 2010, Aachen + + ND survived

3 March 2010, Passau NA NA +, retrospective survived

4 June 2012, Regensburg + + ND died

5 June 2012, Regensburg + + ? survived

6 June 2012, Berlin - + > 6 weeks + survived

7 September 2012; Berlin - + + first indicator survived

Objectives of Our Study

1. Serological pilot study to discover probably not detected anthrax cases in heroin users

2. Genotyping of isolates from 2000 – 2012 to make a comparative analysis

3. Conclusions on the duration and probable intensity of the outbreak event

Serology Pilot Study

Two serum collections:

1.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

2.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)

Serology Study – Clinical Symptoms

Soft tissue symptoms Number (n=288)) PercentageDRUCK

sera collectionCorrectional facility

sera collectionTotal

Compartment

syndrome

24 0 24 8.3

Fasciitis 10 0 10 3.5

Abscesses n. a. 12 12 4.2

Swelling 47 4 51 17.7

Redness (inflammation) 102 1 103 35.8

Fever 28 3 31 10.8

Other 4 1 5 1.7

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

Serology Study

Results Screening ELISA

ELISA(n)

Western blot tested

(n)

Positive by Western blot

(n)

Negative 258 9 0

Borderline 30 30 0

Positive 0 0 0

Total 288 39 0

Table: Summary of serological results, serum collection DRUCK and serum collection correctional facilities, Germany 2010–2011

No difference between both serum collections

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

OD

492

dilution of serum sample

Testing of Patient A-149 on anthrax toxines

PA LF EF

Right places; right persons?

Anthrax Strains for Molecular Investigations

DesignationYear of

isolationSource

A112a, A112b a 2009 Friedrich-Loeffler-Institute Jena

A138 2010 Friedrich-Loeffler-Institute Jena

A294 2012 Statens Serum Institute Copenhagen

A306 2012 Statens Serum Institute Copenhagen

A303 b 2012 Charité Berlin / Robert Koch-Institute

A315/1, A315/2 c 2000 Norwegian Institute of Public Health Oslo

Norway/1983 1983 Norwegian Veterinary Institute Oslo

a A112a and A112b refer to different colony morphologies of the same isolateb only DNA from clinical sample (skin swab) availablec two 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.

Molecular Identification

DesignationYear of isolation

pXO1 pXO2 Dhp61 BA_5345

A112a, 2009 + + +

A112b a 2009 + + +

A138 2010 + + +

A294 2012 + + +

A306 2012 + + +

A303 2012 + + +

A315/1, c 2000 + + +

A315/2 2000 + + +

Norway/1983 1983 + + +

Molecular Typing MLVA 8

Marker/Year

vrrA vrrB1 vrrB2 vrrC1 vrrC2 CG3 pXO1 pXO2

1983 313 229 162 613 604 158 132 139

2000 313 229 162 613 604 158 132 141

2009/2010 313 229 162 613 604 158 132 137

2012 313 229 162 613 604 158 132 139

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

Molecular Typing MLVA 31

MLVA-MarkerA315/1, A315/2

(2000)A112a (2009)

A112b (2009)

A138 (2010)

A294, A303, A306 (2012)

Bams 1 422 422 422 422 422Bams 3 609 609 609 609 609Bams 5 307 307 307 307 307Bams 13 454 454 454 454 454Bams 15 616 616 616 616 616Bams 21 676 676 676 676 676Bams 22 735 735 735 735 735Bams 23 651 651 651 651 651Bams 24 595 595 595 595 595Bams 25 376 376 376 376 376Bams 28 493 493 493 493 493Bams 30 889 862 889 889 889Bams 31 772 772 772 772 772Bams 34 425 425 425 425 425Bams 44 417 417 417 417 417Bams 51 493 493 493 493 493Bams 53 236 236 236 236 236CG3 158 158 158 158 158pXO1 135 135 135 135 135pXO2 141 137 137 137 139vrrA 314 314 314 314 314vrrB1 229 229 229 229 229vrrB2 162 162 162 162 162vrrC1 616 616 616 616 616vrrC2 604 604 604 604 604VNTR 12 115 115 115 115 115VNTR 16 273 273 273 273 273VNTR 17 386 386 386 386 386VNTR 19-2 99 99 99 99 99VNTR 23 197 197 197 197 197VNTR 35 109 109 109 109 109

Expected Fragment Lengths (bp)

High resolution MLVA-31 is suitable for strain analyses

MLVA Results on Bams30 and pXO2-AT

1 A112a and A112b refer to different colony morphologies of the same isolate2 only DNA from clinical sample (skin swab) available

Strain

designation

Year of

isolation

Country Bams30-

repeats

pXO2-AT-

repeats

A315 2000 Norway 75 9

Ba4599 2009 Scotland 75 7

A112a1 2009 Germany 72 7

A112b1 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

A3032 2012 Germany 75 8

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

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.

Clustering of Isolates

Ref.: Grunow R et al. , Euro Surveill. 2013 Mar 28;18(13).

SNP-Analyse

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.

Ref.: Grunow R et al. , Euro Surveill. 2013 Mar 28;18(13).

Full Genome Sequencing of Anthrax Strains

Designation Year of

isolation

Source

1. A112 2009 Friedrich-Loeffler-Institute Jena

[A138 2010 Friedrich-Loeffler-Institute Jena]

2. A294 2012 Statens Serum Institute Copenhagen

3. A306 2012 Statens Serum Institute Copenhagen

[A303 2012 Charité Berlin / Robert Koch-Institute]

4. A315 (1/2) 2000 Norwegian Institute of Public Health Oslo

[Norway/1983 1983 Norwegian Veterinary Institute Oslo]

5. UR-11 2012 University Regensburg

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.

Conclusions (1)

1. Several pathogens could cause anthrax-like clinical manifestations; therefore appropriate laboratory confirmation is required.

2. 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.

3. Serological methods like ELISA and Western blot are important tools for epidemiological investigation and supporting elements for confirmation of diagnoses.

4. 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.

Conclusions (2)

5. Molecular methods for genotyping (MLVA 8/31, SNP-analyses) identified all studied isolates as the same strain, including the Norwegian isolate from 2000.

6. 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.

7. It is not clear how many cases have remained undetected since this time.

8. So far, awareness by physicians and patients is most important for an early and effective treatment.

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

Clusteranlyse anhand der MLVA-31-Profile

The F-complex comprises the eight drug-related isolates.

23