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 2011 (28.3 cases/sentinel) (Fig. 1).
(HFMD) epidemic was experienced since the start of the Clinical specimens were collected from patients with
National Epidemiological Surveillance of Infectious HFMD and herpangina during routine infectious agent
Diseases in Japan (July 1981). Although the most fre­ surveillance between June and November, 2011. All
quent virus detected was coxsackievirus A type 6 samples were transferred to the Kobe Institute of Health
(CVA6), several enteroviruses (EVs) such as CVA16, for laboratory diagnosis.
CVA10, and CVA6 caused the HFMD epidemic in vari­ Specimens were analyzed using consensus­degenerate
ous parts of Japan (1). In Kobe, the epidemic (weekly hybrid oligonucleotide primer (CODEHOP) VP1 RT­
cases per Kobe pediatric sentinel clinic exceeding 1.0) seminested PCR (2–4) to detect EVs. For EV typing, the
started on week 23 and ended on week 38 (a duration of partial VP1 sequences derived from the CODEHOP
16 weeks). The main epidemic peaked at week 28 of products (about 290 base pairs) were determined. The

Fig. 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 of

Health, 4­6 Minatojima­nakamachi, Chuo­ku, Kobe
650­0046, Japan. Tel: {81­78­302­6252, Fax: {81­78­
302­0894, E­mail: kyoko_akiyoshi—office.city.kobe.lg.jp

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resulting sequences were compared with the sequences E6, RD­18S, or/and FL and the other 4 strains from
in the GenBank database of the DNA Data Bank of throat swabs were detected using CODEHOP. CVA10
Japan (DDBJ) using BLAST. Virus isolation was per­ and CVA16 strains were identified using isolates, by
formed using RD­18S, HEp­2, FL, and Vero­E6 cells. neutralization tests with antisera provided by the Na­
Viruses were detected in 59 samples from 53 patients tional Institute of Infectious Diseases or by sequencing
(47 HFMD, 5 herpangina, and 1 exanthema patients) analysis. CVB3 and CVB4 strains were isolated using
between June 2 (week 22) and November 21, 2011 (week FL, HEp­2, and Vero­E6, and identified by neutraliza­
47). The number of patients infected with CVA6, tion tests with antisera (Denka Seiken, Tokyo, Japan).
CVA16, CVA10, CVB3, CVB4, and rhinovirus were 31, Rhinoviruses were detected by CODEHOP.
13, 6, 1, 1, and 1, respectively (Table 1). CVA6 accounted for 82z of viruses detected between
As CVA6 did not grow well in cultured cells, it was weeks 22 and 30, but was not detected after week 31.
directly detected from the clinical specimens using CVA16 was detected from week 22 in the prevalent early
CODEHOP. Though 2 CVA10 strains were isolated us­ stage, while CVA16 accounted for 13z of detected
ing RD­18S, and it was detected by CODEHOP in the viruses between weeks 22 and 30 and was detected spo­
other 5. Ten CVA16 strains were isolated using Vero­ radically until week 47. CVA10 and CVA16 were detect­
ed in 6 and 7 samples, respectively, between weeks 33
Table 1. Viruses detected in the 53 cases and 47. This suggested that the spread of CVA10 and
CVA16 occurred at the same time.
Throat Contents No. of The median ages of patients infected with CVA6,
Disease swab
Stool of blister Saliva cases
CVA16, and CVA10 were 1.8, 2.3, and 3.5 years, re­
CVA6 HFMD 21 3 7 1 29 spectively. Two patients infected with CAV6 were aged
Herpangina 2 2 over 30 years, suggesting that the infection had spread
CVA16 HFMD 13 1 13 to adults.
CVA10 HFMD 2 2 Febrile illness was observed in 84z, 46z, and 83z
Herpangina 3 3 of patients infected with CVA6, CVA16, and CVA10,
Exanthema 1 1 1 respectively. No aseptic meningitis case was found.
CVB3 HFMD 1 1 1 CVA6­positive patients also showed the characteristic
CVB4 HFMD 1 1 skin symptom of large blisters. Some CVA6­positive
Rhinovirus HFMD 1 1 patients showed nail shedding, several weeks after infec­
Total 44 6 8 1 53
tion. CVA16­positive patients showed the characteristic
skin symptoms found in HFMD. Half of the CVA10­
CVA6, coxsackievirus A type 6; CVB3, coxsackievirus B type 3; positive patients were diagnosed with herpangina.
HFMD, hand, foot, and mouse disease. Between June and November 2011, 7 patients were af­

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

Date of Virus
Onset onset Week detected Temperature Symptom Remarks

Patient 1 1st 6/19 24 ND 39.29C Large blisters more than 1 cm, and skin of
hand which peeled off
2nd 7/14 28 CVA16 No fever Vesicular exanthema
3rd 11/12 45 CVA10 No fever Many vesiculrar exanthema of the oral
mucosa and peripheral extremities
Patient 2 1st 6/9 23 CVA61) Unknown Unknown The younger brother
developed HFMD on June 4
2nd 7/16 28 CVA16 No fever Conventional HFMD The younger brother
developed the second HFMD
on July 16
Patient 3 1st 6/24 25 ND No fever Many large blisters on skin of buttocks The younger brother
(Brother of developed HFMD on June 19
Patient 1) 2nd 7/20 29 CVA16 No fever Conventional HFMD The younger brother
developed second HFMD on
July 15
Patient 4 1st 7/11 28 ND Fever Large blisters more than 1 cm, and skin of
hand which peeled off
2nd 7/31 30 CVA16 No fever Conventional HFMD
Patient 5 1st 6/28 26 ND 37.09C Severe symptoms of oral mucosa
2nd 8/22 34 CVA16 No fever Many vesiculrar exanthema of palms and
soles, and many aphthae of tongue
Patient 6 1st 6/24 25 CVA6 Fever Blisters which fused and peeled off became
scabs
2nd 10/12 41 CVA10 39.09
C A lot of small rash
Patient 7 1st 7/14 28 ND 39.09
C Small rash on palms and soles without oral
mucosa, and nail shedding 1 month later
2nd 10/10 41 CVA16 39.09
C Severe inflammation of oral mucosa
1):
CVA6 was detected in a specimen of the younger brother obtained on June 6.
ND, not done. Other abbreviations are in Table 1.

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fected by multiple occurrences of HFMD (Table 2). The lance Report (IASR), vol. 33, p. 59–60, 2012 in
first onset in these 7 cases was between weeks 23 and 28. Japanese.
The first specimen was analyzed in only 1 (Patient 6) of
the 7 cases. CVA6 was detected in this patient. The first Conflict of interest None to declare.
specimen 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 REFERENCES
HFMD episode in Patients 1, 3, 4, and 7, characteristic 1. National Institute of Infectious Diseases and Tuberculosis and In­
fectious Diseases Control Division, Ministry of Health, Labour
skin symptoms of large blisters measuring more than 1 and Welfare (2012): Hand, foot and mouth disease in Japan,
cm in diameter and nail shedding several weeks later, 2002–2011. Infect. Agents Surveillance Rep., 33, 55'–56'.
strongly suggested infection with CVA6. 2. Rose, T.M., Schultz, E.R., Henikoff, J.G., et al. (1998): Consen­
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CVA16 and CVA10 were detected in 6 and 1 patients, distantly related sequences. Nucleic Acids Res., 26, 1628–1635.
3. Allan Nix, W., Steven Oberste, M. and Pallansch, M.A. (2006):
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3 times, suggesting that Patient 1 was infected with 3 direct identification of all enterovirus serotypes from original clini­
different viruses (CVA6, CVA16, and CVA10) succes­ cal specimens. J. Clin. Microbiol., 44, 2698–2704.
sively in the season. 4. Nishimura, Y., Umami, R.N., Yoshida, H., et al. (2009): Identifi­
cation of enterovirus serotypes by CODEHOP PCR. Infect.
Agents Surveillance Rep., 30, 12–13 (in Japanese).
This article appeared in the Infectious Agents Surveil­

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