Helge Karch. Institute for Hygiene and National

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1 EHEC Anmerkungen zum aktuellen Ausbruch Workshop Biologische Gefahren Prävention, Reaktion und Wahrnehmung durch die Bevölkerung Helge Karch Institute for Hygiene and National Consulting Laboratory for HUS University Münster

2 Enterohemorrhagic Escherichia coli Diseases Diarrhea Bloody diarrhea Hemolytic uremic syndrome Neurological symptoms Schweiz Med. Wochenschr. 85: 905 (1955) High public health impact Large outbreaks No specific therapy No specific prevention (vaccination) Conrad Gasser

3 Hemolytic uremic syndrom yndrome: Clinic i and histopathology th Clinic Hemolytic anemia (Hb < 10 g/dl, fragmented erythrocytes) Thrombocytopenia (< /ml) Acute renal failure (Serum creatinin ) Histopathology Glomerular thrombotic microangiopathy

4 Meldepflichtige Krankheiten 6IfSG Krankheitsverdacht, Erkrankung und Tod 6 Abs.1 Nr.1 h) Masern i) Meningokokken- Meningitis oder -Sepsis j) Milzbrand k) Poliomyelitis l) Pest m)tollwut n) Typhus abdominalis/ Paratyphus a) Botulismus b) Cholera c) Diphtherie d) Humane spongiforme Enzephalopathie, p außer familiär-hereditäre Formen e) Akute Virushepatitis f) Enteropathisches hämolytisch-urämisches Syndrom (HUS) g) Virusbedingtes hämorrhagisches Fieber Erkrankung und Tod an einer behandlungsbedürftigen Tuberkulose

5 Meldepflichtige Krankheitserreger 7 Namentliche Meldung nach 7 Abs.1 (Labor) 1. Adenoviren; Konjunktivalabstrich 2. Bacillus anthracis 3. Borrelia recurrentis 4. Brucella sp. 5. Campylobacter sp., darmpathogen 6. Chlamydia psittaci 7. Clostridium botulinum oder Toxinnachweis 8. Corynebacterium diphtheriae, Toxin bildend 9. Coxiella burnetii 10. Cryptosporidium parvum 11. Ebolavirus 12. a) EHEC b) E. coli, sonstige darmpathogene St. 13. Francisella tularensis 14. FSME-Virus 15. Gelbfiebervirus 16. Giardia lamblia 17. Haemophilus influenzae; Liquor oder Blut 18. Hantaviren 19. Hepatitis-A-Virus 20. Hepatitis-B-Virus 21. Hepatitis-C-Virus; keine chron. Infektion 22. Hepatitis-D-Virus 23. Hepatitis-E-Virus 24. Influenzaviren; nur direkter Nachweis 25. Lassavirus 26. Legionella sp. 27. Leptospira interrogans 28. Listeria monocytogenes; Blut, Liquor, Neugeborene 29. Marburgvirus 30. Masernvirus 31. Mycobacterium leprae 32. Mycobacterium tuberculosis/africanum, Mycobacterium bovis, Resistenzbestimmung 33. Neisseria i meningitidis; itidi Liquor, Blut, 34. Norwalk-ähnliches Virus; Stuhl 35. Poliovirus 36. Rabiesvirus 37. Rickettsia prowazekii 38. Rotavirus 39. Salmonella Paratyphi; direkte Nachweise 40. Salmonella Typhi; direkte Nachweise 41. Salmonella, sonstige 42. Shigella sp. 43. Trichinella spiralis 44. Vibrio cholerae O 1 und O Yersinia enterocolitica, darmpathogen 46. Yersinia pestis 47. Andere Erreger hämorrhagischer Fieber

6 Meldeweg in Deutschland 1 Mo 2 Di 3 Mi Meldepflichtige Krankheiten dcwerc fferc dfkfafkm ewfdc wdc djföoijfgp dde dsadgrg FJGOJG sdfdgrertg ^sgtbwetrn 2 ^sddgf g SADaS dsdd casdadsd feiteite eccas FFDOKG werotit FGPGdwr2 fgd dfkasürirjt dgoijpjjgijj dsfoj ^spoewe dfsdgfg dfgdg jdgiaergjgrpog ogipgug goipqre gtijpgo revqekfs efwifeherhgfer erpgjpjt ^ffgvwgbw ^safertbnewrt Berichtswoche 4 Do 5 Fr Labor Arzt Innerhalb von 24 Stunden 6 Sa 7 So 8 Mo 9 Di 1. Woche nach Meldung 2. Woche nach Meldung Wöchentlich 10 Mi 11 Do 12 Fr 13 Sa 14 So 15 Mo 16 Di 17 Mi 18 Do 19 Fr 20 Sa 21 So 22 Mo 23 Di 24 Mi 25 Do Innerhalb einer Woche 3. Woche nach Meldung 26 Fr 27 Sa 28 So Gesundheitsamt Landesstelle Robert Koch Institut Epidemiologisches Bulletin

7 Reported EHEC infections and HUS cases in Germany ( ) 2010) EHEC HUS # year Epidemiol. Bull., RKI

8 Outbreaks caused by EHEC O157:H - in Germany Year (mo) Location HUS Source* cases (n) 1988 (09-11) Bavaria 6 Unknown 1995/96 (12-03) Bavaria 28 Teewurst 2002 (03) Münsterland 6 Unknown 2002 (10-12) Bavaria 38 Apple juice Curdled cheese 2006 (04-07) North Germany 18 Unknown 2009 (07-08) North Germany 4 Unknown * Based on case-control studies; SF STEC O157 never isolated from any food or environmental samples Ammon et al., J. Infect. Dis., 1999; Alpers et al., Epidemiol. Infect., 2009; Epid. Bull. RKI, 24/2006; Pollock et al., Emerg. Infect. Dis. 2010; Nielsen et al., Zoonoses and Public Health, 2011

9 Case-fatality ratio in sorbitol- fermenting EHEC O157:H - outbreaks Outbreak in 1995/96, Germany: 3 of 28 HUS-patients died during the acute phase Case-fatality ratio 11% (Ammon et al., J. Inf. Dis., 1999) Outbreak in 2002, Germany: 4 of 38 HUS-patients died Case-fatality ratio 11% (Alpers et al., Epidemiol. Infect., 2009) Children died after 13 (6-31) days. Causes of death: CNS complications

10 SF EHEC O157:H - : Clinical course of the infection Typically 10% 15% of EHEC O157:H7 infections progress to HUS Limited information for SF EHEC O157:H - Germany: 18 patients with SF EHEC O157 associated diarrhea were fully monitored 12 of 18 (66.6%) developed HUS (Alpers et al.,, Epidemiol. Infect. 2009) Scotland: 18 patients with SF EHEC O157 associated diarrhea 8 of 18 patients (44.4%) developed HUS (Pollock et al., Emerg. Infect. Dis. 2010)

11 Epidemiological curve of the outbreak in 2011 Frank et al., N. Engl. J. Med., 2011

12 Diagnostic scheme for EHEC detection Screening Targets Results Interpretation Action STOOL SAMPLE stx+/eae+/rfb O157 +/sfpa- EHEC O157:H7 Enrichment in GN broth IMS enrichment Plating on: SMAC Isolation of CT-SMAC NSF O157+ colonies Enterohemolysin agar Harvesting the whole bacterial growth into PBS PCR screening for the following targets stx 1,2, 2f eae rfb O157 sfpa stx+/eae+/rfb O157 +/sfpa+ SF EHEC O157:H - stx+/eae+/-rfb O157 -/sfpa- stx+/eae-/-rfb O157 -/sfpa- stx-/eae+/rfbo157+/sfpa+ stx-/eae+/rfbo157-/sfpa- Non-O157 EHEC (eae+) Non-O157 EHEC (eae-) SF O157 stx-/eae+ Non-O157 stx-/eae+ Culture -SMAC - CT-SMAC -EHEC-Hly yagar Isolation - Char. phenotypes - Colony blot hybridization Characterization -SLST(gnd) -MLST - flic typing - Virulence genes

13 Identification of the outbreak strain Prerequisites: 1.) HUSEC-collection 2.) Molecular techniques 3.) Stool sample (arrived on = day 1) Day 1

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15 Identification of the outbreak strain Day 2: Strain isolated from stool sample Molecular analysis: - stx 2 PCR - eae PCR - gnd sequencing - flic restriction fragment length polymorphism - Multi Locus Sequence Typing (adk, fumc, gyrb, icd, mdh, pura, reca) Day 1, Day 2

16 flic Restriction Fragment Length Polymorphism M M bp HhaI flic-rflp patterns of EHEC O HUSEC037 (O104:H21), ST: HUSEC041 (O104:H4), ST: EHEC LB M. molecular weight marker (1kb DNA ladder, Invitrogen).

17 Identification of the outbreak strain Day 3: 1.) Molecular analysis completed: HUSEC041 (O104:H4), stx 2 -positive, ST ) Development of a rapid and highly specific detection method 3.) Comparison with other strains from HUSEC- collection 4.) Phenotypic assays Day 1, Day 2, Day 3

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19 O104:H4 ST678 is the outbreak strain Stx titer Lab.-Nr. ST flic stx Eagg aggr pic tere Vero LB Münster 678 H LB Münster 678 H LB Münster 678 H LB Münster 678 H LB Paderborn 678 H LB Paderborn 678 H LB Frankfurt 678 H LB Frankfurt 678 H LB Frankfurt 678 H LB Münster 678 H LB Münster 678 H LB Münster 678 H LB Hamburg 678 H HUSEC041 Köln 678 H HUSEC037 Hagen 672 H dact Frank et al., , Eurosurveillance

20 Phylogeny of HUS-associated EHEC Analysed strains: 524 ( ) HUSEC collection ( Minimum-spanning tree based on MLST allelic profiles of: adk, fumc, gyrb, icd, mdh, pura, reca Mellmann et al., Emerg. Infect. Dis. 2008

21 Prevalence of EHEC serotypes in sporadic cases of HUS in Germany ( ) O157:H7/H- SF O157:H- O26:H11/H- O103:H2/H- O111:H8/H10/H- O145:H25/H28/H- O91:H21 O55:H7/Hnt O104:H21 O104:H4 O128:H2 O121:H19 O113:H21 O157:H7/H- O98:H- (46.4%) Ont:H21/H-/Hnt (13.6%) OR:H2/H11/H- O165:H25 O119:H2 O112:H- O73:H18 SF O157:H- (19.9%) O163:H19 O70:H8 O136:Hnt n = 588 O76:H19 O174:H21 O26:H11/H-

22 Shiga toxin-producing E. coli O104 from humans H-types: H2, H4, H7, H12, H16, H21 HUS association: H2 (Scotland et al., J.Med. Microbiol., 1988) H21 (CDC, Morb. Mortal Wkly Rep., 1995) H4 (Bae et al., Yonsei Med. J., 2006) (Mellmann et al., Emerg. Infect. Dis., 2008) Isolated in 9/2001: Three sisters with diarrhea or HUS in Cologne (HUSEC041)

23 Previous isolations of EHEC O104:H4 from patients Country Year Diagnosis Suspected Reference source Germany 2001 HUS Unknown Mellmann et al., 2008 France 2004 n.a Unknown ECDC/EFSA 2011 Korea 2005 HUS Unknown Bae et al., 2006 Finland 2010 Diarrhea Travel (Egypt) ECDC/EFSA 2011 No isolation of E. coli O104:H4 reported from animals (ECDC/EFSA 2011)

24 Multiplex PCR for the specific detection of EHEC O104:H4 HUSEC037 O104:H21 HUSEC041 O104:H4 Outbreak strain C600 NTC M Test in 3h bp stx2 rfbo104 terd flic H4 200 Day 1, Day 2, Day 3, Day 6

25 Established rapid and specific test for the outbreak strain

26 Real-time multiplex PCR for stx 2, O104-wzy and flic H4 Test in 68 min Melting Peak: O104-wzy 75 C stx2 80 C flic H C EHEC O104:H4: HUSEC041 LB stx2+ HUSEC037 O104:H21 stx2+ HUSEC003 O157:H7 stx2+ Negative control: C600 NTC Day 1, Day 2, Day 3, Day 6, Day 7

27 Aggregative adherence of E. coli O104:H4 outbreak strain to human intestinal epithelial cells (HCT-8) E. coli O104:H4 outbreak isolate Enteroaggregative E. coli 10µm HUSEC041 (E. coli O104:H4) 10µm HUSEC037 (E. coli O104:H21) 10µm 10µm

28 Intestinal pathogenic Escherichia coli Micro-colonies bioclips Biofilm Cytokines, Enterotoxines, EAST1, Pic, Pet, Shet1 EPEC aepec EAEC Guanylatcylase Lysis of vacuoles Movement Proliferation ETEC EIEC STEC/ EHEC/ HUSEC modified from Kaper et al., Nat. Rev. Microbiol., 2004

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30 Comparison of selected virulence genes of E. coli O104:H4 outbreak isolates, HUSEC041 and EAEC Gene Outbreak isolates HUSEC041 EAEC (n=80) EHEC stx iha lpfa O lpfa O ter cluster irp fyua EAEC aata (paa marker) asta (EAST-1) set pic agga (AAF/I) agg3a (AAF/III) aggr aap Bielaszewska et al., Lancet Infect. Dis., 2011

31 Outbreaks linked to seed sprouts Year Pathogen Cases Location Sprout type 1988 S. Saintpaul 143 UK Mung 1994 S. Bovismorbificans 595 Sweden, Finland Alfalfa 1995 S. Stanley 242 USA, Finland Alfalfa 1996 S. Montevideo 500 USA Alfalfa 2010 S. Bareilly 231 UK Mung 1996 EHEC O157:H Japan (Sakai) Radish 1997 EHEC O157:H7 126 Japan Radish 1997 EHEC O157:H7 85 USA Alfalfa 1998 EHEC O157:H7 9 USA Alfalfa, clover 2011 EHEC O104:H4 3801* Germany Bean * Including 834 cases of hemolytic uremic syndrome and 46 deaths (as of June 27, 2011) modified from Pennington, Lancet Infect. Dis., 2011

32 Purified Shiga toxins Müthing et al., Thromb Haemost., 2009

33 Antibodies against Shiga toxins in German children Number of sera ELISA anti-stx detection anti-stx1 anti-stx2 anti-stx1 + 2 Positive 59 (3.8%) 24 (1.6%) 22 (1.4%) 13 (0.8%) Negative 1484 (96.2%) 1519 (98.4%) 1521 (98.6%) 1530 (99.2%) Total 1543 (100%) 1543 (100%) 1543 (100%) 1543 (100%) Stx2-antibodies in adults (19-63 years): 15/213 (7%)

34 Differences between sorbitol-fermenting E. coli O157:H - and EHEC O104:H4 SF EHEC O157 EHEC O104:H4 Genome EHEC (eae+) EAEC/EHEC (eae-) Antibiotics sensitive ESBL producer Motility - + Seasonality Winter Summer Age of patients < 3 yrs 43 (median) Risk for HUS 44% - 66% 20% Transmission routes? Sprouts Reservoir Humans Humans, Bielaszewska et al., Lancet Infect. Dis., 2011; Frank et al., N. Engl. J. Med., 2011

35 Features of EHEC 104:H4 outbreak strain Rare serotype in HUS All strains express significantly ifi higher amounts of Stx2 than HUSEC041 reference strain Aggregative adherence All belong to HUSEC041 clone All are ESBL producer (and AAF/I) p ( ) in contrast to HUSEC041

36 Perspectives Infectious dose Immunity of the population against E. coli O104:H4 Reservoir(s) Antibiotic treatment? Further spread?

37 Acknowledgments Münster Martina Bielaszewska Wenlan Zhang Alexander Mellmann Robin Köck Andreas Bauwens Georg Peters Berlin Lothar Wieler RKI Wernigerode Angelika Fruth Rita Prager Funding German Federal Ministry i of Education and Research, Network Zoonoses