# Presentation: 783935

## Experiences with non-O157 STEC and implications on Public Health Programs 

**FLEMMING SCHEUTZ**
**STATENS  SERUM  INSTITUT**
**The International *****Escherichia***** and *****Klebsiella***** Centre (WHO)**
**Copenhagen**

## Publications “O157”, “non-O157”, VTEC, STEC & Shiga-Like Toxin in Title/Abstract in PubMed (1982 - 2007)

## Non-O157 STEC studies

**	****	****	****K. E. Johnson et al. CID 2006;43**
**16 countries, 1988-2006  (1,402/2,892)****	****48%**
**	****	****	****Range 19%-100%**
**USA, Canada, UK, Germany, Spain, Italy, Czech Republic, Belgium, France, Denmark, Finland, Sweden, Australia, Chile, Argentina & Japan **
**Netherlands, 2006****	****	****80%**
**Australia, 2004****	****	****64% **
**Belgium, 2006****	****	****81%**
**Brazil, 2007****	****	****100%**
**Poland, 2004****	****	****100%**
**Germany, 1998****	****	****88%**

**Notes:**

1.6% in a study testing faecal samples submitted to the diagnostic laboratories of two hospitals in Melbourne using direct plating, a cytotoxicity assay, and both an ezyme immunoassay and PCR for Shiga toxin [Bennett-Wood 2004] 
In a Spanish study, testing faecal samples submitted from inpatients and outpatients of one hospital by PCR, yielded an overall prevalence of 2.5% [Blanco 2004]. 
In one clinical laboratory in Belgium, during 16 years of screening with PCR, 0.9% of samples were STEC-positive, without any trend in time [Pierard 2006]. 

## Non-O157 STEC surveillance27 countries; 18,302 isolates

**% Non-O157 in the Enter-net database**

**CZ**

**GR**

**MT**

**PL**

**SK**

**GB**

**ES**

**GZ**

**NZ**

**IE**

**NL**

**EE**

**SE**

**JP**

**BE**

**FR**

**HU**

**AT**

**FI**

**NO**

**CH**

**IT**

**LU**

**DK**

**DE**

**SI**

**ZA**

**70**

**3**

**20**

**13**

**6**

**5949**

**87**

**1517**

**607**

**642**

**457**

**27**

**510**

**1576**

**314**

**322**

**48**

**312**

**115**

**130**

**18**

**164**

**42**

**872**

**4232**

**246**

**3**

**Country**

## Non-O157 STEC surveillance27 countries; 6,480 isolates

## STEC incidences in Europe

## STEC in Denmark2006

**2**

**0**

**5**

**3**

**32**

**3**

**0**

**5**

**8**

**20**

**No cases**

**1 - 1,49**

**1,5 – 1,99**

**Incidence of STEC **
**(per 100.000)**

**0 - 0,49**

**0,5 - 0,99**

**2 – 3,99**

**13**

**12**

**> 4**

**STEC in Denmark****2006**

**29**

**0**

**1**

**25**

**Number of diagnosed STEC infections by county, and annual incidence of **_**all**_** STEC infections in 2006**

**12 !**

## Detection ratio of STEC in counties using molecular methods vs "other methods" in Denmark

**Detection ratio of STEC in counties using molecular methods vs "other methods" in Denmark**

## Non-O157 STEC outbreaks

**O26:H11**
**O103:H2**
**O103:H25**
**O104:H21**
**O111:H- / H2 / H8****	**
**O113:H21**

**O117:H4**
**O118:H2**
**O119**
**O121:H19 / H21**
**O128:H2**
**O145:H-**
**O?:H19**
***Citrobacter freundii***

**Non-O157 STEC outbreaks**

## O103:H25 outbreak in Norway

**HUS ”outbreak” notified**

**Date of onset of disease**

**HUS cases**
**Diarrhea cases**

## Source: Sliced, dry fermented lamb’s sausage

## Product and environmental samples

**	****	****O103 positive**
**Cured meat products****	****2462****	****53**
**Sheep meat****	****171****	****9**
**Environment****	****296****	****0**
**Spices, additives, culture****	****57****	****0**
**Minced meat****	****1000****	****0**
**All isolates were *****stx2***** negative & *****eae***** pos.**
**BUT clustered with patient isolates by MLVA (DNA fingerprint)**

## O26:H11 outbreak in Denmarkstx1 & eae

**O26:H11 outbreak in Denmark*****stx1 & eae***

**20 cases; all children**
**median 2 years**
**Very mild symptoms**
**Discovered by PFGE**
**Little media attention**

**17 cases; 15 children**
**10 with HUS**
**1 child died **
**HUS notification**
**Massive media attention**

**O103:H25 outbreak in Norway*****stx2 & eae***

## Lessons learned in Norway

**Outbreak ****d****iscovered due to notification of cases of HUS**
**Methods in clinical laboratories were inadeqaute for detection of non-O157 in 5 out of the six first cases of HUS**
***stx2***** negative isolates dominated**
**MLVA was used to identify cases and the source**

## Lessons learned in Denmark

**Real-time PFGE of Danish non-O157 detected a ”mild” outbreak**
**Only possible because isolates were available for typing**
**Source identified using**
**access to purchase records**
**cooperation with supermarkets searching their central computers**

## HUS and STEC notifications worldwide 

**	****7****	****Hungary, Japan, Poland**
**	****	****Notification dates from 1998 – 2005**
**STEC only****	****Austria, Canada****, Estonia, ****Finland, **
**	****13****	****Greece,Iceland, Ireland, Luxembourg, Malta , ****	****	****	****New Zealand, Norway, Slovenia, Sweden **
**	****	****Notification dates from 1990 – 2005**
**Not mandatory****	****England & Wales, France †, Italy ‡,**
**	****7****	****Romania, Spain, Scotland*, South Africa**
**	****	****Pediatric- nephrology network since **
**	****	****† ****1996 < 15 Yrs****	****	****‡ 1988 < 14 Yrs**
**	****	*****Laboratory based since mid-eight’ies**

**HUS and STEC notifications worldwide**** **

## stx2 OR* 32.5 > stx2c OR* 4.7 for HUS

| Subtype | Non-HUS * | HUS* |
| --- | --- | --- |
| stx2 | 60 | 11 |
| stx2c | 49 | 1 |
| stx2d-activatable | 4 |  |
| stx2d | 39 |  |
| stx2e | 2 |  |
| stx2-variant | 3 |  |
| stx2 + stx2c | 23 | 7 |
| stx2 + stx2d | 1 |  |
| 2x stx2-activatable | 4 |  |
| stx2c + stx2-activatable | 1 |  |
| Total | 186 | 19 |

**Ethelberg et al. 2004 EID; 10**

**Shiga toxin 2 (*****stx2*****) subtype and clinical presentation**

***stx2***** OR* 32.5 > *****stx2c***** OR* 4.7 for HUS**
***) OR: odds ratio; multivariant analysis adjusted for age**

## Virulence profile and clinical manifestation in 559 Danish STEC patients 1994-2005 

**0%**

**20%**

**40%**

**60%**

**80%**

**100%**

***stx2 +***

***eae***

***stx1 +***

***stx2 +***

***eae***

***stx1 +***

***eae***

***stx2***

***stx1 +***

***stx2***

***stx1***

**Other**

**D**

**PD**

**BD**

**PBD**

**HUS**

## Stx1 : 4 subtypes a - d7-8 variants

**d**

**b**

**c**

**a**

- Stx1a-S._dysenteriae-3818T

- Stx1a-S._sonnei-CB7888

- Stx1b-O111-CB168

- Stx1b-O157-EDL933

- Stx1b-O48-94C

- Stx1b-O111-PH

- Stx1c-O174-DG131-3

- Stx1d-ONT-MHI813

## Stx2 :7 subtypesa - g35 variants

**d**

**f**

**b**

**g**

**c**

**a**

**e**

**Stx2 :****7 subtypes****a**** - ****g****35 variants**

## Slide 21

## Only 12 Stx2 variants found in Danish patients

**Stx2a-O48-94C**

** **

**Stx2a-O157-EDL933**

** **

** **

_**Stx2c-O157-FLY16**_

** **

** **

**Stx2d-O8-C466-01B**

** **

**Stx2d-O157-7279**

**Stx2e-ONT****-26725-97**

** **

_**Stx2g-O2-7v**_

**Stx2b-O118-EH250**

**0.01**

**Stx2c-O157-C394-03**

**Stx2d-O73-C165-02**

**Stx2e-O101-E43**

**Stx2b-O111-PH**

**New variants**
**First time in humans**

## Attack rate of Stx2 variants associated with HUS

**O157**
**Stx2a-O157-EDL933 + Stx2c-O157-FLY16****	****6/23****	****26%**
**Stx2a-O157-EDL933****	****3/17****	****18%**
**Stx2c-O157-FLY16****	****1/18****	****6%**
**Stx2a-O157-SF + Stx2c-O157-FLY16** **	****1/1****	****-**
**Non-O157**
**Stx2a-O48-94C****	****6/20****	****30%**
**Stx1b + Stx2a-O157-EDL933****	****1/3****	****-**
**Stx2a-O157-EDL933 +** **Stx2c-O157-FLY16****	****1/2****	****-**
**Stx1b + Stx2a-O48-94C****	****1/5****	****20%**

## Conclusions

**Two Stx2a***** *****variants associated with HUS**
**Stx2a-O157-EDL933****	****in NSF O157**
**	****(& Stx2-O157-FLY16)**** ****	**
**Stx2a-O157-SF ****	****in SF O157**
**Stx2a-O48-94C****	****in ****Non-O157**
**New Paradigm:**
**How may STEC be classified?**
**Are certain virulence ”cocktails” associated with severe disease rather than the serotype?**

## Classification of STEC in 5 Sero-pathotypes

**Classification of STEC in 5 Sero-pathotypes**
**Karmali *****et al.***** , 2003, J. Clin. Microbiol., 41:4930-40**

**Sero-**
**pathotype**

**Relative **
**incidence **

**Frequency **
**of **
**involvement **
**in outbreaks**

**Association **
**with severe **
**disease **
**(HUS or HC)**

**Serotypes**

**A**

**High**

**Common**

**Yes**

**O157:H7, O157:NM**

**B**

**Moderate**

**Uncommon**

**Yes**

**O26:H11, O103:H2, **
**O111:NM, O121:H19,**
**O145:NM**

**C**

**Low**

**Rare**

**Yes**

**O91:H21, O113: H21, **
**O104:H21, others**

**D**

**Low**

**Rare**

**No**

**multiple**

**E**

**Non human only **

** **

** **

**multiple**

**New Paradigm**

## Problems with this classification

**Association with serotype and not with virulence profile**
**More than 120 O:H serotypes have been associated with**
**	****HUS (Bergey’s Manual of Systematic Bacteriology, 2nd ed.)**
**Many O:H serotypes display extensive heterogeneity**
**Involvement in outbreak may rapidly change**
**Relative incidence **
**is scewed by lack of effecient detection methods**
**will vary depending on the epidemiology of specific types**

**New Paradigm**

## 1.	HUS inducing STEC and/or an epidemic outbreak potential
eae and stx2a
eae  negative and stx2d (activatable)
eae  and stx1  	Less common but certain O:H serotypes have 	been associated with HUS

Diarrhea inducing in humans! 
Many different virulence profiles
Capacity to produce Stx and association with diarrhea in humans

3.	Animal-associated STEC

High prevalence in the animal reservoir
Seems to be their natural habitat 
No human cases
Candidates for this group are stx2e positives

***eae***** and *****stx2a***
***eae*****  negative and *****stx2d***** (activatable)**
***eae*****  and *****stx1*****  ****	****Less common but certain O:H serotypes have ****	****been associated with HUS**
**Diarrhea inducing in humans!**** **
**Many different virulence profiles**
**Capacity to produce Stx and association with diarrhea in humans**
**3.****	****Animal-associated STEC**
**	**
**High prevalence in the animal reservoir**
**Seems to be their natural habitat **
**No human cases**
**Candidates for this group are *****stx2e***** positives**

**Alternative classification**

**New Paradigm**

## Questions

**Should management and treatment of patients be adjusted according to virulence cocktail?**

**New Paradigm**

## Danish Practice since 2000

**ALL patients with STEC are excluded**
**or quarantined if they are**
**Children in institutions and day care**
**Staff of health care facilities**
**Hospital staff or hospitalized patients**
**Food handlers**

**Background**

**and until they have had two consecutive STEC negative stool samples **
**Prolonged shedding of STEC has resulted in huge social problems especially for parents**

## Danish example of consequences

**Revision of guidelines for treatment of Danish patients with STEC may include antibiotic treatment of asymptomatic patients with **
***  ******eae***** negative STEC**
***  ******eae***** & *****stx1***** ****	****- except some serotypes **
**Asymptomatic patients are likely to be allowed back in institutions and day care after treatment**

**New Paradigm**

## Adequate detection methods should include the isolation of bacteria

Typing methods should be standardized

Subtyping methods for Stx2a variants associated with HUS should be implemented

Urgent need for standardized nomenclature

**Typing methods should be standardized**
**Subtyping methods for Stx2a***** *****variants associated with HUS should be implemented**
**Urgent need for standardized nomenclature**

**Recommendations**

## Questions

**How much is detection and surveillance scewed?**
**Can case definitions for HUS to be notified within the Public Health system be established?**
**Will management and treatment of STEC patients depend on**
**a case-to-case based assessment? **
**an outbreak-to-outbreak approach?**
**local epidemiology?**

## YES - some

**Should non-O157:H7 STECs be considered to be adulterants as *****E. coli***** O157:H7? **

**YES - some**

## Acknowledgements

**Statens Serum Institut, Copenhagen**
**Joan Neverman Jensen **
**Søren Persson**
**Katharina E. P. Olsen**
**Co-authors on nomenclature**
**Lothar Beutin, ****Federal Institute for Risk Assessment, Berlin**
**Denis Piérard, ****Academisch Ziekenhuis Vrije Universiteit, Brussels**
**Nancy A. Strockbine, ****National Center for Zoonotic, Vector-borne and Enteric Diseases (CDC), Atlanta**** **