Jpn. J. Infect. Dis., 65, 459­461, 2012

Laboratory and Epidemiology Communications

Enteroviruses in Patients Experiencing Multiple Episodes of Hand,
Foot, and Mouth Disease in the Same Season in Kobe, Japan, 2011
Kyoko Akiyoshi*, Tomoko Suga, and Ai Mori
Kobe Institute of Health, Kobe 650­0046, Japan
Communicated by Takaji Wakita(Accepted July 20, 2012)
In 2011, the largest hand, foot, and mouth disease(HFMD) epidemic was experienced since the start of theNational Epidemiological Surveillance of Infectious
Diseases in Japan (July 1981). Although the most fre­quent virus detected was coxsackievirus A type 6(CVA6), several enteroviruses (EVs) such as CVA16,CVA10, and CVA6 caused the HFMD epidemic in vari­
ous parts of Japan (1). In Kobe, the epidemic (weeklycases per Kobe pediatric sentinel clinic exceeding 1.0)
started on week 23 and ended on week 38 (a duration of16 weeks). The main epidemic peaked at week 28 of2011 (28.3 cases/sentinel) (Fig. 1).
Clinical specimens were collected from patients with
HFMD and herpangina during routine infectious agent
surveillance between June and November, 2011. All
samples were transferred to the Kobe Institute of Healthfor laboratory diagnosis.
Specimens were analyzed using consensus­degenerate
hybrid oligonucleotide primer (CODEHOP) VP1 RT­
seminested PCR (24) to detect EVs. For EV typing, thepartial VP1 sequences derived from the CODEHOP
products (about 290 base pairs) were determined. TheFig. 1. Weekly cases of hand, foot, and mouth disease (HFMD) per pediatric sentinel clinic, and incidences of infec­
tion with viruses among HFMD, herpangina, and exanthema patients in Kobe during 2011.

*Corresponding author: Mailing address: Kobe Institute ofHealth, 4­6 Minatojima­nakamachi, Chuo­ku, Kobe
650­0046, Japan. Tel: {81­78­302­6252, Fax: {81­78­302­0894, E­mail: kyoko_akiyoshioffice.city.kobe.lg.jp459

resulting sequences were compared with the sequencesin the GenBank database of the DNA Data Bank ofJapan (DDBJ) using BLAST. Virus isolation was per­formed using RD­18S, HEp­2, FL, and Vero­E6 cells.
Viruses were detected in 59 samples from 53 patients(47 HFMD, 5 herpangina, and 1 exanthema patients)
between June 2 (week 22) and November 21, 2011 (week47). The number of patients infected with CVA6,CVA16, CVA10, CVB3, CVB4, and rhinovirus were 31,
13, 6, 1, 1, and 1, respectively (Table 1).
As CVA6 did not grow well in cultured cells, it wasdirectly detected from the clinical specimens using
CODEHOP. Though 2 CVA10 strains were isolated us­
ing RD­18S, and it was detected by CODEHOP in theother 5. Ten CVA16 strains were isolated using Vero­

E6, RD­18S, or/and FL and the other 4 strains fromthroat swabs were detected using CODEHOP. CVA10and CVA16 strains were identified using isolates, byneutralization tests with antisera provided by the Na­tional Institute of Infectious Diseases or by sequencing
analysis. CVB3 and CVB4 strains were isolated usingFL, HEp­2, and Vero­E6, and identified by neutraliza­tion tests with antisera (Denka Seiken, Tokyo, Japan).
Rhinoviruses were detected by CODEHOP.
CVA6 accounted for 82z of viruses detected between
weeks 22 and 30, but was not detected after week 31.
CVA16 was detected from week 22 in the prevalent earlystage, while CVA16 accounted for 13z of detectedviruses between weeks 22 and 30 and was detected spo­radically until week 47. CVA10 and CVA16 were detect­
ed in 6 and 7 samples, respectively, between weeks 33
and 47. This suggested that the spread of CVA10 andCVA16 occurred at the same time.
The median ages of patients infected with CVA6,
CVA16, and CVA10 were 1.8, 2.3, and 3.5 years, re­spectively. Two patients infected with CAV6 were aged
over 30 years, suggesting that the infection had spreadto adults.
Febrile illness was observed in 84z, 46z, and 83z
of patients infected with CVA6, CVA16, and CVA10,
respectively. No aseptic meningitis case was found.
CVA6­positive patients also showed the characteristic
skin symptom of large blisters. Some CVA6­positivepatients showed nail shedding, several weeks after infec­
tion. CVA16­positive patients showed the characteristic
skin symptoms found in HFMD. Half of the CVA10­
positive patients were diagnosed with herpangina.
Between June and November 2011, 7 patients were af­

Table 1. Viruses detected in the 53 cases
No. ofContents
ThroatStool of blister Saliva cases
swab

DiseaseCVA6

HFMD
Herpangina
CVA16
HFMD
CVA10
HFMD
Herpangina
Exanthema
CVB3
HFMD
CVB4
HFMD
Rhinovirus HFMD

212
13
2
3
1
1
1Total

44

3

7

1

29
2
13
2
3
1
1
1
1

153

1

1
1
1
6

8

CVA6, coxsackievirus A type 6; CVB3, coxsackievirus B type 3;
HFMD, hand, foot, and mouse disease.

Table 2. Cases with multiple episodes of hand, foot, and mouth disease in Kobe, Japan, 2011
Onset

Date ofonset

Week

1st6/1924

2nd3rd7/14
11/12

1stVirus
detected

Temperature

Symptom

ND

39.29
C28
45

CVA16
CVA10

No feverNo fever

6/923

CVA61)

Unknown

Large blisters more than 1 cm, and skin ofhand which peeled off
Vesicular exanthema
Many vesiculrar exanthema of the oral
mucosa and peripheral extremities
Unknown

2nd7/1628

CVA16

No fever

Conventional HFMD

Patient 3(Brother ofPatient 1)

1st6/24

25

ND

No feverMany large blisters on skin of buttocks

2nd7/20

29

CVA16

No fever

Conventional HFMD

Patient 4

1st7/1128

NDFever

Patient 5

2nd
1st
2nd7/31
6/28
8/22

30
26
34

CVA16
ND
CVA16

No fever37.09
CNo fever

Patient 6

1st6/24

25

CVA6Fever

Patient 7

2nd
1st10/12
7/14

41
28

CVA10
ND

39.09
C39.09
C

2nd

10/10

41

CVA16

39.09
C

Large blisters more than 1 cm, and skin ofhand which peeled off
Conventional HFMD
Severe symptoms of oral mucosa
Many vesiculrar exanthema of palms andsoles, and many aphthae of tongue
Blisters which fused and peeled off becamescabs
A lot of small rash
Small rash on palms and soles without oral
mucosa, and nail shedding 1 month later
Severe inflammation of oral mucosa

Patient 1

Patient 21): CVA6 was detected in a specimen of the younger brother obtained on June 6.

ND, not done. Other abbreviations are in Table 1.

460

Remarks

The younger brotherdeveloped HFMD on June 4
The younger brotherdeveloped the second HFMD
on July 16
The younger brotherdeveloped HFMD on June 19
The younger brotherdeveloped second HFMD onJuly 15

fected by multiple occurrences of HFMD (Table 2). The
first onset in these 7 cases was between weeks 23 and 28.
The first specimen was analyzed in only 1 (Patient 6) ofthe 7 cases. CVA6 was detected in this patient. The firstspecimen from Patient 2 was not analyzed; however,CVA6 was detected in the younger brother of Patient 2
who developed HFMD at the same time. In the first
HFMD episode in Patients 1, 3, 4, and 7, characteristic
skin symptoms of large blisters measuring more than 1
cm in diameter and nail shedding several weeks later,strongly suggested infection with CVA6.
At the second HFMD episode in the 7 patients,CVA16 and CVA10 were detected in 6 and 1 patients,respectively. Furthermore, Patient 1 developed HFMD
3 times, suggesting that Patient 1 was infected with 3
different viruses (CVA6, CVA16, and CVA10) succes­
sively in the season.

lance Report (IASR), vol. 33, p. 5960, 2012 inJapanese.
Conflict of interest None to declare.
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This article appeared in the Infectious Agents Surveil­

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