A verocytotoxin-producing E. coli outbreak with a
surprisingly high risk of haemolytic uraemic syndrome,
Denmark, September-October 2012
B Soborg (firstname.lastname@example.org)1,2, S G Lassen1, L Müller1, T Jensen3, S Ethelberg1,4, K Mølbak1, F Scheutz4
1. Department of Infectious Disease Epidemiology, Statens Serum Institut, Copenhagen, Denmark
2. European Programme for Intervention Epidemiology Training (EPIET), European Centre for Disease Prevention and Control,
(ECDC), Stockholm, Sweden
3. The Danish Veterinary and Food Administration, Copenhagen, Denmark
4. Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark
Citation style for this article:
Soborg B, Lassen SG, Müller L, Jensen T, Ethelberg S, Mølbak K, Scheutz F. A verocytotoxin-producing E. coli outbreak with a surprisingly high risk of haemolytic
uraemic syndrome, Denmark, September-October 2012 . Euro Surveill. 2013;18(2):pii=20350. Available online: http://www.eurosurveillance.org/ViewArticle.
Article submitted on 17 December 2012 / published on 10 January 2013
Denmark faced an outbreak of verocytotoxin-produc-
ing E. coli (VTEC) O157:H7 infections in autumn 2012.
Thirteen cases were diagnosed of which eight had
haemolytic uraemic syndrome (HUS). Epidemiological
investigations suggested ground beef as the vehicle
of the outbreak. The outbreak strain had a rare toxin
gene subtype profile: eae, vtx1a and vtx2a, and a high
proportion of HUS (62%) among cases, a finding previ-
ously linked with the outbreak subtype profile. Toxin
subtyping can be useful to identify high risk VTEC
In late September 2012, a paediatric department in a
Central Copenhagen Hospital notified Statens Serum
Institut (SSI) of four cases of haemolytic uraemic syn-
drome (HUS). This led to the initiation of an outbreak
HUS and infections with verocytotoxin-producing
E. coli (VTEC) are individually notifiable in Denmark.
Furthermore, VTEC infections are laboratory-notifiable.
Around 180 cases are reported each year, of which
serogroup O157 constitute 15–20% . Previously,
Denmark has experienced only two general outbreaks
of VTEC infection. In 2006 an outbreak of VTEC O157
affected 25 cases, with organic pasteurised milk as the
source . In 2007, an outbreak of VTEC O26 affected
20 cases, with an organic fermented beef sausage as
the source . In addition, Denmark reported 26 cases
during the large VTEC O104 outbreak in Germany in
2011 . The annual number of HUS cases in Denmark
ranges from two to six . Subtyping of verocytotoxin
genes are done at SSI as described in .
For this outbreak, we defined a confirmed case as
an individual with a laboratory-confirmed infection
with VTEC O157 eae, vtx1a and vtx2a diagnosed after
1 August 2012, or a person diagnosed with HUS in
the same time period and a serology-confirmed VTEC
O157 infection. A probable case was a person without
laboratory-confirmed VTEC infection, diagnosed with
HUS after 1 August 2012, or a person diagnosed with
VTEC O157 infection in the same period, but without
subtyping, or a person diagnosed with VTEC eae, vtx1a
and vtx2a infection in the same period, but without
O-typing. The date 1 August was chosen in order to
identify any early cases.
On 22 October, after laboratory confirmation of VTEC
O157 in two patients, SSI notified Danish clinical labo-
ratories of a possible VTEC outbreak to increase case
ascertainment and reporting. We interviewed patients
(or their parents for children under 15 years of age) by
telephone, using a structured trawling questionnaire.
Early investigations had pointed to ground beef as
the possible outbreak source, and information on pur-
chase dates and other possible labelling information of
ground beef was collected and presented to the super-
markets for further tracing of possible batch identifi-
cation. The Danish Veterinary and Food Administration
traced back the ground beef.
SSI performed live-slide agglutination on the submit-
ted isolates upon arrival, and all VTEC O157-positive
isolates were typed immediately with the PCR protocol
for subtyping of vtx genes . Further testing included
conventional (and confirmatory) O:H serotyping, Vero
cell assay, fermentation of sorbitol, production of beta-
glucuronidase, presence of additional virulence genes
by dot blot hybridisation and/or PCR, and pulsed-field
gel electrophoresis (PFGE). We asked patients who
were culture-negative for VTEC O157 to submit a serum
specimen for serology testing for antibodies against
We identified 13 cases of VTEC O157 (11 confirmed
and two probable). Eight cases had HUS. The cases
belonged to nine families. Eleven reported symptoms,
whereas two were diagnosed when we screened
healthy family members. Date of disease onset ranged
from 18 September to 28 October 2012 (Figure). The
cases were distributed throughout the country, eight
were female, and the median age was 14 years (range:
Hypothesis-generating interviews suggested that the
source of the outbreak was food served in the house-
holds. Most affected families had young children, ate
traditional (non-vegetarian) Danish food and lived in
provincial towns. The most frequent food exposure
reported was ground beef: all cases had eaten ground
beef during the incubation period, six in the form of
pan-fried ground beef (“hakkebøffer”). Two house-
holds mentioned the beef still being red in the centre
when eaten. No other food items were common to all
cases. The families had purchased food in a variety of
different supermarket chains. Trace-back of the beef
indicated that it was of Danish origin and narrowed
the possibilities down to two major Danish slaugh-
terhouses and to two consecutive slaughter days.
However, identification of a single food producer was
impossible and no further action was taken.
We compared interview information from the current
outbreak with that of a salmonella outbreak occur-
ring in 2011 in the same months (to account for pos-
sible changes in eating habits with the seasons) ,
where a comparable hypothesis-generating question-
naire was used. The nine families involved in the 2012
VTEC outbreak reported similar frequencies of con-
sumption as the 2011 salmonella cases for almost all
food items. However, all nine case-families in the 2012
VTEC outbreak reported eating ground beef, compared
with 14 of 24 case-families in the salmonella outbreak
(p=0.03; Fisher’s exact-test).
The outbreak strain was a typical VTEC O157:H7, eae
positive, non-sorbitol fermenting, negative for beta-
glucuronidase. However, it had a rare toxin subtype
profile including the genes vtx1a and vtx2a. All strains
had the same PFGE profile, which had not been reported
previously among Danish VTEC isolates. The Table
shows the distribution of the total number of VTEC
O157 cases diagnosed and sub-typed in Denmark from
1997 to 2012, stratified by toxin profile. For each toxin
profile, the proportion of cases with HUS is shown.
VTEC infections containing the vtx2a toxin profile were
associated with a higher number of HUS cases (from
3% to above 20%), and VTEC infections containing the
eae + vtx1a + vtx2a profile were associated with even
higher frequencies of HUS cases (33%).
The present outbreak represents the first food-borne
outbreak of a highly virulent VTEC O157 in Denmark.
Subtyping of the toxin genes allowed rapid identifica-
tion and classification of cases. In addition, toxin sub-
typing was of relevance for the overall risk assessment.
The combination of vtx1a and vtx2a in the toxin profile
of the outbreak strain is rare and appears to be asso-
ciated with a high risk of progression to HUS as also
indicated by previously subtyped VTEC O157 strains.
This emphasises that within the VTEC O157 group,
there is a diversity of strains with different potential of
As a surrogate for an analytical epidemiological inves-
tigation, we compared case exposure to ground beef
Probable and confirmed cases of symptomatic verocytotoxin-producing E. coli O157 by date of onset, Denmark,
September –October 2012 (n=11)
The two asymptomatic cases found by screening are not depicted in the figure.
3839 4041 4243 44
X Haemolytic uraemic syndrome
Week of symptom onset
Number of cases
Confirmed case (n=9) Probable case (n=2)
3www.eurosurveillance.org Download full-text
with similar exposure in a recent salmonella outbreak.
This case¬–case comparison suggested that more
families than expected had consumed ground beef in
the present outbreak. In addition, these families had
prepared it in a way that allowed for part of the beef
to remain uncooked. This suggests that this outbreak
was caused by ground beef, a common source of VTEC
outbreaks. Ground beef has a short shelf life (seven
days), which may have limited the size of this outbreak.
If the vehicle of the outbreak had been a food item with
longer shelf life, the public health impact of this high
risk HUS-associated VTEC outbreak could have been
Based on the findings of this outbreak we suggest per-
forming toxin subtyping in similar outbreak situations
in order to quickly identify high risk VTEC strains and
thereby aid risk assessment of the outbreak.
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Verocytotoxin-producing E. coli O157 cases and
proportion of haemolytic uraemic syndrome by toxin
profile, Denmark, 1 January 1997–31 July 2012 (n=212)
HUS: haemolytic uraemic syndrome.
eae + vtx1a
eae + vtx1a + vtx2c
eae + vtx2c
eae + vtx2a + vtx2c
eae + vtx2a
eae + vtx1a + vtx2a