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Published Date: 2008-02-18 20:00:19
Subject: PRO/AH/EDR> E. coli VTEC non-O157, past. ice cream, 2007 - Belgium: Antwerp
Archive Number: 20080218.0655

E. COLI VTEC NON-O157, PASTEURIZED ICE CREAM, 2007 - BELGIUM: (ANTWERP)
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Date: Thu 14 Feb 2008
Source: Eurosurveillance Surveillance and Outbreak reports, 2008
13(7) [edited] <http://www.eurosurveillance.org/edition/v13n07/080214_5.asp>

In October 2007, an outbreak of verocytotoxin-producing _Escherichia
coli_ (VTEC) O145 and _E. coli_ O26 occurred among consumers of ice
cream produced and sold in September 2007 at a farm in the province
of Antwerp (Belgium). The ice cream was consumed at 2 birthday
parties and also eaten at the farm. A total of 5 children, aged
between 2 and 11 years, developed hemolytic uremic syndrome (HUS),
and 7 other co-exposed persons contracted severe diarrhea. In 3 of
the 5 HUS cases, VTEC O145 infections were laboratory confirmed, one
in association with VTEC O26. Identical isolates of _E. coli_ O145
and O26 were detected with PCR (polymerase chain reaction) and PFGE
(pulsed field gel electrophoresis) in fecal samples of patients and
in ice cream leftovers from one of the birthday parties, in fecal
samples taken from calves, and in samples of soiled straw from the
farm at which the ice cream was produced. Ice cream was made from
pasteurized milk and most likely contaminated by one of the food handlers.

Introduction
------------
Verocytotoxin-producing _Escherichia coli_ (VTEC), including _E.
coli_ O157:H7, O26, O145, and other _E. coli_ serotypes, are
important causes of gastrointestinal illness and hemolytic uremic
syndrome (HUS) in young children. This syndrome is characterized by
hemolytic anemia, thrombocytopenia, and acute renal failure, a
complication occurring in 5-14 percent of VTEC infections (1,2). HUS
is a potential life-threatening disease and can induce hypertension,
proteinuria, and chronic renal failure in 5 percent of affected
patients. The age group primarily affected is children under 5 years.
VTEC O157:H7 is considered as the most clinical significant serotype,
and is often associated with severe bloody diarrhea and HUS. The
prevalence of VTEC serotype O145 in human infections is relatively
low, accounting for 5-7 percent of all non-O157 strains in prevalence
studies (3). The range of products associated with VTEC infections is
wide: hamburger, ground beef, cider, spinach, unpasteurized ice
cream, milk and cheese, and others. Infections have also been linked
to municipal water supplies (4,5).

VTEC infections in Europe and the USA have increased in the last
decade causing several large epidemics of food poisoning in
industrialized countries (4-6). In Belgium, however, it has been a
sporadic disease (7,8). In 2005, the incidence of VTEC in Belgium was
0.5 cases per 100 000 population, compared to a mean incidence in
Europe of 1.2 cases per 100 000 population (7-9). However, this is
probably an underestimation, as most of the country's clinical
laboratories do not test for these microorganisms in routine
gastroenteritis samples (8).

We report on 5 children with HUS of which 3 had a laboratory
confirmed VTEC O145 infection. All patients consumed ice cream
produced and sold at a farm.

Methods
-------
The outbreak:
On 2 Oct 2007, the detection of 3 isolates of VTEC O145, 1 of which
was associated with _E. coli_ O26, was reported by Belgium's Federal
Reference Laboratory for _E. coli_ to the Antwerp department of
Infectious Diseases Control. The strains were obtained from patients
hospitalized with HUS and living in the northern part of Antwerp
province. On 3 Oct 2007, investigators instituted active case finding
and interviewed the parents of the different patients. All patients
had eaten ice cream produced and sold at the same farm within 8 days
of developing gastrointestinal symptoms. The ice cream was consumed
at 2 birthday parties or consumed on the farm.

The farm was a traditional dairy farm with a limited number of cows,
young cows and calves. The farm that made the ice cream was well
known in the region and, depending on season and weather, up to 160
(42.25 gal) liters of ice cream were sold daily.

On 3 Oct 2007, the Antwerp Department of Control of Infectious
Diseases invited the Antwerp Department of the Federal Agency for the
Safety of the Food Chain, the Laboratory of Food Microbiology of the
University of Ghent, and the Reference Laboratory for _E. coli_ of
the University of Brussels to assist in the investigation. The study
was carried out to determine the impact of the outbreak, to identify
risk factors, and to interrupt transmission. On 5 Oct 2007,
investigators were informed of the existence of leftovers of ice
cream consumed at one of the birthday parties.

Epidemiologic investigation:
In order to develop hypotheses regarding possible sources of _E.
coli_ O145 infections investigators interviewed the parents of the
patients who contracted HUS on 3 Oct 2007 using an adapted standard
questionnaire for HUS' investigation generated by the Netherlands'
Landelijke Coordinatiestructuur Infectieziektebestrijding
(Coordination Structure for Combating Infectious Diseases) (10). They
were asked about consumption of food and drinks, contact with animals
(domestic, farm, zoo), and travel history in the 10 days before onset
of diarrhea.

Hypothesis-generating interviews suggested that the outbreak occurred
among participants in 2 birthday parties with 8 and 11 participants
respectively, respectively on 14 and 16 Sep 2007. A 3rd group of
consumers was an undefined group of individuals who consumed ice
cream at the farm. Consequently a retrospective cohort study was
conducted among the participants in the birthday parties.

For case-ascertainment purposes, a probable case of HUS was defined
as a patient who developed acute diarrhea (3 or more loose stools in
a 24-hour period) complicated with HUS (acute hemolytic anemia,
thrombocytopenia, and signs of renal failure) occurring within 10
days of consumption of ice cream produced at a farm in Mol in
September 2007. A confirmed case with HUS was defined as a patient
meeting the criteria of a probable case and accompanied by isolation
of _E. coli_ O145 and/or _E. coli_ O26 in stools or a positive
serology for _E. coli_ O145 and/or O26. A patient with a probable
VTEC diarrhea infection without HUS was defined as a patient who
developed acute diarrhea (3 or more loose stools in a 24-hour period)
in the 10 days following consumption of the farm-made ice cream and
the patient belonged to a group in which a confirmed case has been
detected. A patient with only a confirmed VTEC infection was a
patient meeting the criteria of a probable VTEC diarrhea infection
accompanied by _E. coli_ O145 and/or _E. coli_ O26 in stools or a
positive serology for E. coli O145 and/or O26.

A retrospective cohort study was established among the participants
in the birthday parties. Relative risks and P-values (Fisher exact)
were calculated using Epi Info, version 3.3.2 (11).

Environmental investigation:
The farm's layout, ice cream production process, and staff activities
were determined. Different environmental samples were obtained: fecal
samples from animals (calves, young animals, and cows), and samples
from each pen floor, dust, and feed. To identify the source of the
infection, raw milk, fresh ice cream produced at the farm, and
leftover portions of the ice cream from the birthday party on 16 Sep
2007 were sampled.

Microbiological examination and molecular analysis:
Stools and urine samples of HUS patients were collected, and sent to
the Belgian reference laboratory for _E. coli_ for microbiological
analysis. The stools were cultured using SMAC/SMAC+CT medium. On the
basis of biochemical tests, PCR, and agglutination assay VTEC of
serogroup O145 and O26 were identified. Additional PCR tests were
performed to identify specific virulence genes carried by these VTEC
strains. Serum samples of the 5th HUS patient were collected and
tested for presence of anti-VTEC antibodies using agglutination assay.

PFGE was used to examine and to compare the genetic profiles of the
VTEC isolates.

Results
-------
Clinical information and epidemiologic information:
By 5 Oct 2007, 5 cases of HUS had been identified among consumers of
ice cream sold at the farm between 12 and 16 Sep 2007 (Figure I -
[see original URL - Mod.LL]). All patients with HUS were girls aged
between 2 and 11 years; 3 cases met the criteria of a confirmed case.
VTEC of serogroups O145 and O26 were isolated from feces and urine in
a 2-year-old girl and serogroup O145 was isolated from the feces of
an 8-year-old girl. In a 3rd case, serology was positive for O145
antigen. All HUS patients were admitted to the hospital, 2 requiring
hemodialysis, and 3 transfusion. No deaths have occurred among the
identified patients

Of the cases, 2 had eaten ice cream at the farm and 3 during birthday
parties. Among persons co-exposed at the same birthday parties, 7
cases of acute diarrhea were identified. The age of the diarrhea
patients ranged from 4 to 61 years.

The distribution of the HUS cases and the VTEC diarrhea by time and
place of consumption of the ice cream is shown in Figures 1 and 2
[see original URL - Mod.LL]. The mean incubation period between
infection and onset of diarrhea was 5 days. The mean interval between
onset of diarrhea and HUS was 5.6 days.

Of all the VTEC cases, only 2 patients had visited the farm but had
had no contact with the stables or animals. No cases of VTEC diarrhea
could be identified among the consumers of ice cream at the farm.

The attack rate for patients with HUS among the participants in
Birthday Party 1 on 14 Sep 2007 was 12.5 percent (1/8) and 18 percent
(2/11) in Birthday Party 2 on 16 Sep 2007. The attack rate for the
probable VTEC infections was 25 percent (2/8) in Birthday Party 1 and
73 percent (8/11) in Birthday Party 2. No diarrheal illness was
reported among the farm workers and the staff involved in the ice
cream preparation. Relative risks were undefined in Birthday Party 1
and 2. P-values calculated with Fisher exact one tailed test were
p=0.75 in Birthday Party 1 and p=0.20 in Birthday Party 2.

Environmental study and microbiologic data:
Evaluation of the ice cream production process did not reveal major
processing errors. Pasteurized milk was used for the production of
the ice cream. One person who was normally not involved in the
production process of ice cream and who also worked at the farm
participated only in the production of ice cream in the week of 12
Sep 2007. Fresh milk stored at the farm and prepared ice cream
samples collected on 4 Oct 2007 were negative for VTEC O145 and O26
pathogenic bacteria, but fecal samples of calves and dust samples of
the calves' stables were positive for VTEC O145 and O26. Leftovers of
the ice cream consumed at Birthday Party 2 on 16 Sep 2007 were also
positive for VTEC O145 and O26.

Molecular analysis:
Sorbitol-fermenting VTEC O145 strains were identified in stools of 2
HUS patients, one in association with serogroup O26 that was isolated
in both stool and urine samples. PCR analysis revealed that the VTEC
O145 and O26 isolates were positive for, respectively, verocytotoxin
type 2 and type 1 (VT2 and VT1). Both serogroups were positive for
additional virulence genes eaeA and ehxA. Agglutination assay
performed on the serum samples of the 5th HUS patient revealed the
presence of anti-VTEC O145 antibodies.

PFGE was performed on the VTEC strains isolated from patients, ice
cream, and the farm environment. These results confirmed that the
VTEC O145 strains, isolated from the 2 female patients were
undistinguishable from isolates from ice cream and samples collected
on the farm (Figure 3.A, lanes 3, 4, 6, 7, 8, and 9. The VTEC O26,
isolated from feces and urine of a 2-year-old female patient, were
undistinguishable from VTEC O26 isolated from the environment of the
farm and the ice cream (Figure 3.B lanes 3, 4, 6, and 7) [for
figures, see original URL. - Mod.LL]

Discussion
----------
We have described an outbreak of VTEC infections among individuals
who had eaten ice cream produced and sold at a farm in the northern
part of the province of Antwerp. The infections were transmitted from
animals and their environment to people.

There is strong epidemiological evidence to assume that the
incriminated vehicle was contaminated ice cream. All patients had
eaten ice cream sold at the farm in the week before onset of the
diarrhea and HUS. Individuals participating in the birthday parties
where the ice cream was eaten and who had never been at the farm,
developed the disease. Most likely due to the small number of
participants in the parties, a significant association among the ice
cream eaters and the disease could not be identified. The leftovers
of the ice cream and the stools of the 2 patients with positive
cultures had identical PFGE profiles for VTEC O145 and O26
respectively. Taking into account that pasteurized milk was used in
the production of the ice cream, cross-contamination is the most
likely explanation for the contamination. One of the most likely
explanations might be the participation in the production process of
an individual who was not trained, properly instructed and had
contact with the animals.

Only the 5 HUS cases were tested for non-O157 VTEC and of these only
3 were confirmed as VTEC infections. The VTEC diarrhea cases were not
confirmed by laboratory testing. Testing of fecal samples of patients
with diarrhea for the presence of VTEC is not regularly undertaken in
Belgium. One of the reasons might be that testing for _E. coli_ is
not reimbursed by social security in Belgium. The delay between acute
diarrhea and the onset of HUS, late diagnosis, and the intake of
antibiotics before diagnosis might also explain the absence of VTEC
confirmation in 2 of the HUS patients.

To our knowledge, this is the 1st outbreak of HUS and VTEC caused by
O145 and O26 in Belgium associated with the consumption of ice cream
made from pasteurized milk. Outbreaks associated with VTEC O157 among
visitors to a dairy farm were recently described in Belgium (12),
with consumption of unpasteurized milk as a source of the VTEC, as
described by Allerberger et al (13).

There is significant morbidity and mortality associated with
diarrhea-associated HUS in children due to the devastating
microvascular thrombotic angiopathy (1,2). A Canadian prospective
study showed an annual incidence of 1.11 case of diarrhea associated
HUS per 100 000 children under the age of 16 years (14). The disease
occurred most frequently in children younger than 5 years old (15).
However, in this outbreak, only one out of 5 patients belonged to
this age group. This probably underscores the underdetection of HUS
in the population.

Fecal samples of calves and dust of the barn were positive for VTEC
O145 and O26. Studies on prevalence of _E. coli_ 0157 in cattle in
Belgium show percentages ranging from 0 to 85 percent according to
age of animals, specific farms, herds, and time of sampling. Young
animals in particular have higher carriage. The prevalence of _E.
coli_ O157:H7 in beef carcasses was 1.1 percent (N=2554) in 2005,
while no VTEC were detected in 175 samples of raw milk in 2005 (7).
No data are yet available regarding the prevalence of other
serogroups in Belgian cattle.

This outbreak underscores the need to consider zoonotic transmission
and to highlight the prevention measures in facilities where there is
easy contact with farm animals and their environment. Moreover, in
our case the presence of VTEC in cattle at the farm and the shared
activities of food handling are problematic, as these pathogens can
survive for months on surfaces (3).

The association between ice cream made with pasteurized milk and VTEC
is very unusual (13,14). However as shown in this outbreak,
cross-contamination is a significant risk. Our data underline the
need to reinforce hygienic measures for food-handlers working at
farms where food products are prepared.

This study illustrates the usefulness of appropriate source tracing
in VTEC infections and possibilities of good collaboration among the
clinicians, microbiologists, and public health officials.

Control measures
----------------
To control the outbreak GPs [general practicioners], pediatricians,
hospitals, and health authorities were alerted and asked to look for
cases. The food-preparing process and the quality of the ice cream
and milk were checked and identified as free of VTEC. The food
handlers were informed about the risk of contamination and
prevention. Finally, the Safety in the Food Chain Agency is
considering launching a prevention campaign targeted at this kind of facility.

References
----------
1. Tarr PI, Gordon CA, Chandler WL: Shiga-toxin-producing
_Escherichia coli_ and haemolytic uraemic syndrome. Lancet. 2005; 365: 1073-86.
2. Boyce TG, Swerdlow DL, Griffin PM: _Escherichia coli_ O157:H7 and
the haemolytic-uremic syndrome. N Engl J Med 1995: 333: 364-8.
3. Busch U, Hormansdorfer S, Schranner S, et al: Enterohemorrhagic
_Escherichia coli_ Excretion by Child and her Cat. Emerg Infect Dis
2007; 13: 348-9.
4. European centre for disease control and prevention: Annual
epidemiological report on communicable diseases in Europe. December
2007: 34-9. Available from
<http://www.ecdc.europa.eu/pdf/ECDC_epi_report_2007.pdf>
5. Maki DG: Don't Eat Spinach -- Controlling Foodborne Infections. N
Engl J Med 2006 ;355: 1952-5.
6. Crump JA, Sulka AC, Langer AJ, et al: An outbreak of _Escherichia
coli_ O157:H7 infections among visitors to a dairy farm. N Engl J Med
2002; 347: 555-60.
7. Working group on Foodborne Infections and Intoxications. Verotoxin
producing _Escherichia coli_. In: Trends and sources report on
zoonotic agents in Belgium in 2005. Eds: FAVV-AFSCA. Brussels:
Federal Agency for the safety of the food chain 2007: 61-5.
8. Pierard D, De Zutter L, Cobbaut K, Lauwers S: Enterohemorrhagische
_Escherichia coli_ 0157 en andere serotypes: voorkomen in Belgie bij
mens, dier en levensmiddelen. In: 22e Seminarie: Diagnose en
surveillance van infectieuze aandoeningen 2006. Ed: Wetenschappelijk
instituut Volksgezondheid. Brussels: ISP 2006: 7-13.
9. De Valk H: Epidemie van infecties met _Escherichia coli_ O157:H7
veroorzaakt door de consumptie van rundergehakt, Frankrijk 2005. In:
Diagnose en surveillance van infectieuze aandoeningen 2006. Eds.:
Wetenschappelijk instituut Volksgezondheid. Brussels: ISP 2006:17-20.
10. Steenbergen J, Timen A: _E. coli_ infectie. In: Guidelines
Infectious Disease Control for the Netherlands 2006. Bilthoven: RIVM
2006: 111-6.
11. Centers for Disease Control and Prevention: Epi Info Version 3.3.2, 2005.
12. Van den Branden D, De Schrijver K, Evens K, et al: Een cluster
van _E. coli_ O157:H7 infecties met een hemolytisch uremisch syndroom
na een verblijf op een vakantiehoeve. Vlaams Infectieziektebulletin
2007; 60: 3-10.
13. Allerberger F, Wagner M, Schweiger P, et al: _Escherichia coli_
O157 infections and unpasteurised milk. Euro Surveill. 2001; 6(10):
147-151. Available from
<http://www.eurosurveillance.org/em/v06n10/0610-222.asp>
14. Proulx F, Sockett P: Prospective surveillance of Canadian
children with the haemolytic uraemic syndrome. Pediatr Nephrol. 2005;
20: 786-90.
15. Haeghebaert S, Vaillant V, Decludt B, Grimont PAD: Surveillance
of haemolytic uraemic syndrome in children under 15 years of age in
France in 1998. Euro Surveill. 2000; 5(6): 68-73. Available from
<http://www.eurosurveillance.org/em/v05n06/0506-222.asp>

[Reported by: De Schrijver K (<koen.deschrijver@wvg.vlaanderen.be>)/1
, /2, Buvens G /3, Posse B /4, Van den Branden D /1, Oosterlynck C
/5, De Zutter L /4, Eilers K /1, D Pirard D /3, Dierick K /6, Van
Damme-Lombaerts R /5, Lauwers C /7, Jacobs R /7

/1 Department of Control of Infectious Diseases, Antwerp, Belgium
/2 Department of Epidemiology and Social Medicine, University of
Antwerp, Belgium
/3 Belgian Reference Laboratory for E. coli Department of
Microbiology, University of Brussels, Belgium
/4 Laboratory for Food Microbiology, Department of Veterinary Public
Health and Food Safety University of Ghent, Ghent, Belgium
/5 Department of Paediatric Nephrology, Catholic University of
Leuven, Leuven, Belgium
/6 Department of Microbiology, Institute of Public Health, Brussels, Belgium
/7 Federal Agency for the Safety of the Food Chain, Antwerp, Belgium]

--
Communicated by:
ProMED-mail
<promed@promedmail.org>

[This well documented report is important for several reasons
including the percentage of affected individuals who developed HUS,
the dual nature of non-O157 serotypes, and the implication of a
pasteurized dairy product as the vehicle. The ice cream was
presumably contaminated after pasteurization although it is not clear
that the pasteurization process for that lot was adequate.

ProMED-mail has previously posted an outbreak of this disease from
Eurosurveillance, which was linked to pasteurized milk (E. coli
O157, pasteurized milk - Denmark (Copenhagen) 20040513.1297).

Although much attention has been paid to O157:H7 strains of _E. coli_
that (by virtue of toxin production) cause enterohemorrhagic disease
with or without hemolytic-uremic syndrome (HUS), other serotypes of
_E. coli_ have been associated with this illness as well.

_E. coli_ strains can be grouped by the presence of their O (somatic)
and H (flagellar) antigens, hence O157:H7. The toxins produced are
generally one or 2 Shiga toxins (stx1 and stx2) as well as eae, a
protein intimin that is responsible for attachment of the organism
and mucosal effacing lesions and other virulence factors including
E-hly, espA, etp, and katP.

Other _E. coli_ serogroups that have been associated with VTEC
(verotoxin-producing _E. coli_) disease include motile ones such as
O26:H11 and O104:H21 and non-motile ones such as O111:NM (or H-).
Such non-O157 isolates can be obtained from sheep and cattle and
although they cause as many as 30 percent of outbreaks of VTEC (1),
appear to be somewhat less (or at least more variably) virulent in a
variety of in vivo and in vitro assays (2-4). In analyzing the
genetic and phenotypic profiles of non-O157 groups, it has been found
that they belong to their own lineages and have unique profiles of
virulence traits different from O157 (5). The serogroups appearing to
be most prominent are O26, O111, O128, and O103 (6), the former
serotype being the implicated strain in this outbreak.

If a laboratory is using sorbitol-MacConkey (sMAC) plates to identify
VTEC by virtue of O157's inability to ferment sorbitol, the non-O157
strains will be missed. In a 3-year pediatric study from the
University of Washington, USA (7), 1851 stool samples were processed
for sorbitol fermentation as well as toxin production by EIA (enzyme
immunoassay), and 28 strains of O157 were found along with O103 (4
strains), O118 (2 strains), O111 (2 strains), and 3 other strains.

Clinically, the O157 infections had a higher frequency of bloody
stools, fecal leukocytes, and abdominal pain with shorter symptom
duration. Five (18 percent) of O157 infections developed HUS; none of
the non-O157 strains did. Since toxin assay did not identify all O157
strains found on sMAC plates, the investigators did not advocate
performing toxin assay alone. Non-O157 can produce hemolytic-uremic
syndrome, as demonstrated by a cluster of O121 cases associated with
a lake in Connecticut, USA (8).

Since toxin assays are not uniformly performed in many areas, and
most cases do not produce HUS, it is likely that cases due to
non-O157 strains are being missed. How frequent this phenomenon will
become over time is unclear.

References
----------
1. Hussain HS, Omaye ST: Introduction to the food safety concerns of
verotoxin-producing _Escherichia coli_. Exp Biol Med 2003; 228: 331-2.
2. Blanco J, Blanco M, Blanco JE, et al: Verotoxin-producing
_Escherichia coli_ in Spain: prevalence, serotypes, and virulence
genes of O157:H7 and non-O157 VTEC in ruminants, raw beef products,
and humans. Exp Biol Med 2003; 228: 345-51.
3. Law D, Kelly J: Use of heme and hemoglobin by _Escherichia coli_
O157 and other Shiga-toxin-producing _E. coli_ serogroups. Infect
Immun 1995; 63: 700-2.
4. Tzipori S, Wachsmuth KI, Smithers J, Jackson C: Studies in
gnotobiotic piglets on non-O157:H7 _Escherichia coli_ serotypes
isolated from patients with hemorrhagic colitis. Gastroenterology
1988; 94: 590-7.
5. Schmidt H, Geitz C, Tarr PI, et al: Non-O157:H7 pathogenic
Shiga-toxin producing _Escherichia coli_: phenotypic and genetic
profiling of virulence traits and evidence for clonality. J Infect
Dis 1999; 179: 115-23.
6. Bettelheim KA: Role of non-O157 VTEC. Symp Ser Soc Appl Microbiol
2000; (29): 38-50S.
7. Klein EJ, Stapp JR, Calusen CR, et al: Shiga toxin-producing
_Escherichia coli_ in children with diarrhea: a prospective
point-of-care study. J Pediatr 2002; 141: 172-7.
8. McCarthy TA, Barrett NL, Hadler JL, et al: Hemolytic-uremic
syndrome and _Escherichia coli_ O121 at a lake in Connecticut, 1999.
Pediatrics 2001; 108: E59.

A map of Belgium showing the location of Antwerp in the Flanders
region can be found at
<http://www.state.gov/cms_images/belgium_map_2007-worldfactbook.jpg>- Mod.LL]

See Also