Jpn. J. Infect. Dis., 58, 2005

Laboratory and Epidemiology Communications

An Outbreak of Mixed Infection of Enterohemorrhagic Escherichia coli
O26:H11 and Norovirus Genogroup II at a Kindergarten in Shimane, Japan
Setsuko Iizuka*, Yoshie Tsunomori, Kenji Tabara, Kazuo Tsuda1 and Tsuneo Fukuma2
Shimane Prefectural Institute of Public Health and Environmental Science, Shimane 690-0122,
Oki Public Health Center, Shimane 685-8601 and 2Matsue Public Health Center, Shimane 690-0882, Japan

1

Communicated by Haruo Watanabe(Accepted August 22, 2005)
On January 17, 2005, a health center in Shimane Prefecture received a report that 21 of 72 children in a kindergarten
were absent due to diarrhea and vomiting. Investigation by
the health center revealed that absenteeism due to the above
symptoms began to increase on January 14. A total of 28
kindergarten children got sick in the following 4 days. Someof the family members of the affected kindergarten children
showed the same symptoms. However, 12 members of thekindergarten staff were unaffected.
The non-synchronous appearance of the patients suggested
viral or bacterial infections. Real-time PCR (1) detected
norovirus genogroup II (NV GII) in the stool specimens of
14 of the 16 kindergarten children examined and in those of
6 of 6 family members examined. Bacteriological investigation of the stool specimens of these patients and those ofan additional one kindergarten child and 4 kindergarten staff(total 27 individuals) revealed that 6 kindergarten children
and 3 family members were infected with enterohemorrhagic
Escherichia coli (EHEC) O26:H11(VT1-positive). Therefore,
we further examined the stool specimens of the remaining
kindergarten children, staff members and family members(a total of 88 individuals), and found that one symptomatic
infant and 2 asymptomatic family members were infectedwith EHEC O26:H11(VT1). Bacteriological investigation of
the remaining food samples and the swab specimens of thekindergarten facility gave negative results.
Figure 1 shows the time course of the appearance ofpatients classified according to the pathogens detected. The
patients defined here are those having diarrhea or vomiting
later than January 8, when the first symptomatic case withboth NV GII and EHEC O26:H11(VT1) infections appeared.
The patients appeared on January 15 - 17 in a cluster. Therewas no correlation between the onset of the symptoms andinfections by EHEC O26:H11(VT1) and/or NV GII. TheEHEC O26:H11(VT1)-infected individuals tended to havefever more often.
Table 1 shows the summary data classified according to
the 5 classes by age, with class 1 being the youngest andclasses 4 and 5 the oldest. The symptomatic patients appearedin all the classes at variable frequencies. There were 11 casesof family infection. In all families except one, infection ofthe kindergarten children preceded that of the other family(A)Case

9876543210Jan. 81012141618≧20(B)Case

109876543210O26(NT), NV(NT)O26(–), NV(NT)O26(–), NV(+)
O26(+), NV(NT)O26(+), NV(+)

Jan. 81012141618≧20Fig. 1. Time course of appearance of patients.
(A) Kindergarten children. (B) Family members. NT, not done; –, notdetected; +, detected.

members, suggesting transmission from the kindergarten
children to other family members.
For 12 of the EHEC O26:H11(VT1) isolates tested, thepulsed-field gel electrophoresis (PFGE) of XbaI-digested
chromosomal DNA gave the same pattern (data not shown;
PFGE was conducted by the National Institute of Infectious
Diseases). For all the NV GII isolates, the PCR products
amplified by using the G2SKF/R primer (2) were of the same
single strand conformation polymorphism (SSCP) pattern(data not shown), and the nucleotide sequence of the PCR
products was that of genotype GII/4 (3). These data suggested*Corresponding author: Mailing address: Shimane Prefectural Institute of Public Health and Environmental Science, Nishihamasadacho 582-1, Matsue, Shimane 690-0122, Japan. Tel: +81-85236-8188, Fax: +81-852-36-8356, E-mail: iizuka-setsuko@pref.
shimane.lg.jp329

Jpn. J. Infect. Dis., 58, 2005
Table 1. Detection of EHEC O26:H11 (VT1) and NV GII among different groups, kindergarten children,
kindergarten staff and family members
No. of persons

Symptomatic patients

O26:H11(VT1) positive

NV GII positive

101126121312–

85118
3020

4 (2)*1 (1)1 (1)01 (1)
05 (3)

4351
10655

12 (8)

20Class 1 (0 - 1 y)Class 2 (1 - 3 y)Class 3 (3 - 4 y)Class 4 (5 - 6 y)Class 5 (4 - 6 y)
Staff of the kindergarten
Family members
Total*: Numbers in parentheses show positive for both EHEC O26:H11(VT1) and NVII.

Fukushi, S., Hoshino, F. B., Takeda, N. and Katayama,
K. (2003): Broadly reactive and highly sensitive assay
for Norwalk-like viruses based on real-time quantitative
reverse transcription-PCR. J. Clin. Microbiol., 41, 15481557.
2. Kojima, S., Kageyama, T., Fukushi, S., Hoshino, F. B.,Shinohara, M., Uchida, K., Natori, K., Takeda, N. andKatayama, K. (2002): Genogroup-specific PCR primers
for detection of Norwalk-like viruses. J. Virol. Methods,
100, 107-114.
3. Kageyama, T., Shinohara, M., Uchida, K., Fukushi, S.,
Hoshino, F. B., Kojima, S., Takai, R., Oka, T., Takeda,
N. and Katayama, K. (2004): Coexistence of multiple
genotypes, including newly identified genotypes, in outbreaks of gastroenteritis due to Norovirus in Japan. J.
Clin. Microbiol., 42, 2988-2995.

that the outbreak was caused by a single strain of each ofEHEC O26:H11 (VT1) and NV GII.
The infection source could not be identified, but thekindergarten was undoubtedly the place of the spread of thepathogens. The outbreak pattern and clinical pictures reported
here were indistinguishable from those caused by NV GII
alone. The double infection was revealed only by the simultaneous virological and bacterial examinations.
We thank Prof. Yoshikura, Emeritus Researcher of theNational Institute of Infectious Diseases, for advice on
preparing the manuscript.
This article appeared in the Infectious Agents Surveillance
Report, vol. 26 no. 6, p. 147-148, 2005 in Japanese.
REFERENCES
1. Kageyama, T., Kojima, S., Shinohara, M., Uchida, K.,330