RESEARCH ARTICLE

Effectiveness of Trivalent InactivatedInfluenza Vaccine in Children Estimated by a
Test-Negative Case-Control Design Study
Based on Influenza Rapid Diagnostic Test
Results
Masayoshi Shinjoh1,2☯, Norio Sugaya3☯*, Yoshio Yamaguchi4‡, Yuka Tomidokoro5‡,
Shinichiro Sekiguchi1‡, Keiko Mitamura6‡, Motoko Fujino7‡, Hiroyuki Shiro8‡,
Osamu Komiyama9‡, Nobuhiko Taguchi3‡, Yuji Nakata10‡, Naoko Yoshida11‡,
Atsushi Narabayashi12‡, Michiko Myokai13‡, Masanori Sato14¤a‡, Munehiro Furuichi15¤b‡,
Hiroaki Baba16‡, Hisayo Fujita17‡, Akihiro Sato18‡, Ichiro Ookawara19‡,
Kenichiro Tsunematsu20‡, Makoto Yoshida21‡, Mio Kono22‡, Fumie Tanaka23‡,
Chiharu Kawakami24, Takahisa Kimiya5, Takao Takahashi1, Satoshi Iwata2,25, Keio
Pediatric Influenza Research Group¶
OPEN ACCESS
Citation: Shinjoh M, Sugaya N, Yamaguchi Y,
Tomidokoro Y, Sekiguchi S, Mitamura K, et al. (2015)
Effectiveness of Trivalent Inactivated Influenza
Vaccine in Children Estimated by a Test-Negative
Case-Control Design Study Based on Influenza
Rapid Diagnostic Test Results. PLoS ONE 10(8):e0136539. doi:10.1371/journal.pone.0136539
Editor: Jodie McVernon, Melbourne School of
Population Health, AUSTRALIA
Received: February 3, 2015
Accepted: August 5, 2015
Published: August 28, 2015
Copyright: © 2015 Shinjoh et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source arecredited.
Data Availability Statement: All relevant data arepresented within the paper.
Funding: The authors received no specific funding
for this work.
Competing Interests: Within 36 months, MS has
received speakers' honoraria from Chugai, Shionogi,
Daiichi Sankyo, Japan Vaccine, and Astellas; andgrant support from Japan Vaccine outside of thesubmitted work. NS has received speakers' honoraria
from Chugai, Shionogi, Daiichi Sankyo,

1 Department of Pediatrics, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan, 2 Center for
Infectious Diseases and Infection Control, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan,
3 Department of Paediatrics, Keiyu Hospital, Yokohama, Kanagawa, Japan, 4 Department of ClinicalResearch, National Hospital Organization, Utsunomiya, Tochigi Medical Center, Tochigi, Japan,
5 Department of Pediatrics, Tokyo Metropolitan Ohtsuka Hospital, Toshima-ku, Tokyo, Japan, 6 Departmentof Pediatrics, Eiju General Hospital, Taito-ku, Tokyo, Japan, 7 Department of Pediatrics, Saiseikai Central
Hospital, Minato-ku, Tokyo, Japan, 8 Department of Pediatrics, Yokohama Rosai Hospital, Yokohama,
Kanagawa, Japan, 9 Department of Pediatrics, National Hospital Organization, Tokyo Medical Center,
Meguro-ku, Tokyo, Japan, 10 Department of Pediatrics, Nippon Kokan Hospital, Kawasaki, Kanagawa,Japan, 11 Department of Pediatrics, Kyosai Tachikawa Hospital, Tachikawa, Tokyo, Japan, 12 Departmentof Paediatrics, Kawasaki Municipal Hospital, Kawasaki, Kanagawa, Japan, 13 Department of Pediatrics,
Shizuoka City Shimizu Hospital, Shizuoka, Shizuoka, Japan, 14 Department of Pediatrics, Tokyo Dental
College Ichikawa General Hospital, Ichikawa, Chiba, Japan, 15 Department of Pediatrics, Saitama City
Hospital, Saitama, Saitama, Japan, 16 Department of Pediatrics, Fuji Heavy Industries Health InsuranceSociety Ota Memorial Hospital, Ota, Gunma, Japan, 17 Department of Pediatrics, Hiratsuka Kyosai Hospital,
Hiratsuka, Kanagawa, Japan, 18 Department of Pediatrics, Yokohama Municipal Citizen's hospital,
Yokohama, Kanagawa, Japan, 19 Department of Pediatrics, Japanese Red Cross Shizuoka Hospital,
Shizuoka, Shizuoka, Japan, 20 Department of Pediatrics, Hino Municipal Hospital, Hino, Tokyo, Japan,
21 Department of Pediatrics, Sano Kousei General Hospital, Sano, Tochigi, Japan, 22 Department ofPediatrics, National Hospital Organization Saitama National Hospital, Wako, Saitama, Japan,
23 Department of Pediatrics, Hiratsuka City Hospital, Hiratsuka, Kanagawa, Japan, 24 Yokohama City
Institute of Health, Yokohama, Kanagawa, Japan, 25 Department of Infectious Diseases, Keio University
School of Medicine, Shinjuku-ku, Tokyo, Japan☯ These authors contributed equally to this work.
¤a Current Address: Department of Pediatric Intensive Care, Nagano Children’s hospital, Azumino, Nagano,Japan¤b Current Address: Division of Infectious Diseases, Department of Medical Subspecialties, National Centerfor Child Health and Development, Setagaya-ku, Tokyo, Japan‡ These authors also contributed equally to this work.
¶ Membership of the Keio Pediatric Influenza Research Group is listed in the Acknowledgments.
* sugaya-n@za2.so-net.ne.jp

PLOS ONE | DOI:10.1371/journal.pone.0136539 August 28, 2015

1 / 15Effectiveness of Trivalent Inactivated Influenza Vaccine in Children

GlaxoSmithKline, Astellas, Takeda, Mitsubishi
Tanabe, and Denka Seiken. YY has received
speaker's honorarium from Shionogi. KM has
received speakers' honoraria from Shionogi, Daiichi
Sankyo, Japan Vaccine, Takeda, Astellas, and Denka
Seiken. TT has received speakers' honoraria fromJapan Vaccine and Astellas; and grant support from
Chugai and Shionogi outside of the submitted work.
SI has received speakers' honoraria from Chugai,
Shionogi, Daiichi Sankyo, GlaxoSmithKline, Japan
Vaccine, Astellas, Takeda, and Denka Seiken; andgrant support from Chugai, Shionogi, Daiichi Sankyo,
GlaxoSmithKline, Japan Vaccine, Takeda, andAstellas outside of the submitted work. Other authors
have no competing interests regarding this study.
This does not alter the authors' adherence to PLOS
ONE policies on sharing data and materials.

Abstract
We assessed vaccine effectiveness (VE) against medically attended, laboratory-confirmed
influenza in children 6 months to 15 years of age in 22 hospitals in Japan during the 2013–14 season. Our study was conducted according to a test-negative case-control design
based on influenza rapid diagnostic test (IRDT) results. Outpatients who came to our clinics
with a fever of 38°C or over and had undergone an IRDT were enrolled in this study.
Patients with positive IRDT results were recorded as cases, and patients with negative
results were recorded as controls. Between November 2013 and March 2014, a total of
4727 pediatric patients (6 months to 15 years of age) were enrolled: 876 were positive for
influenza A, 66 for A(H1N1)pdm09 and in the other 810 the subtype was unknown; 1405
were positive for influenza B; and 2445 were negative for influenza. Overall VE was 46%(95% confidence interval [CI], 39–52). Adjusted VE against influenza A, influenza A(H1N1)
pdm09, and influenza B was 63% (95% CI, 56–69), 77% (95% CI, 59–87), and 26% (95%CI, 14–36), respectively. Influenza vaccine was not effective against either influenza A or
influenza B in infants 6 to 11 months of age. Two doses of influenza vaccine provided better
protection against influenza A infection than a single dose did. VE against hospitalizationinfluenza A infection was 76%. Influenza vaccine was effective against influenza A, especially against influenza A(H1N1)pdm09, but was much less effective against influenza B.

Introduction
Influenza vaccination is the most effective method of preventing influenza virus infection andits potentially severe complications, and vaccine efficacy from randomized control trials [1–3]and vaccine effectiveness (VE) from observational studies [4–7] in healthy children has beenreported to be 40%-70%. However, VE varies considerably with time, place, and degree of antigenic distance between the vaccine strain and circulating strain. Although VE has generally
been interpreted in the context of vaccine matches with circulating strains, low VE has recently
been reported to be related to mutations in the egg-adapted H3N2 vaccine strain rather than toantigenic drift in circulating viruses [8,9].
Low VE against influenza A/H3N2 was reported during the 2012–13 season, especially inthe elderly (-11% and 9%) [10,11], even though the vaccine and epidemic strains matched. By
contrast, during the 2013–2014 season, influenza A(H1N1)pdm09 virus caused major epidemics in the United States (U.S.) and Canada, and high VE (60% to 75%) against influenza A(H1N1)pdm09 virus was reported [12,13]. The vaccine and epidemic strains also matched in
the 2013–2014 season. However, since there was a marked change in VE between the 2012–2013 season and 2013–2014 season because of the difference in epidemic influenza subtypes, it
has become very important to estimate VE every season to monitor the performance of current
influenza vaccine.
Although in many countries influenza vaccination is recommended starting at 6 months ofage, there are few studies evaluating the effectiveness of trivalent inactivated influenza vaccine(TIV) in young children. TIV has been reported not to reduce the influenza A infection attack
rate in 6–24 month-old children [14]. By contrast, a recent study revealed TIV was highly effective in children aged <24 months [15].
Indirect protection of the elderly by influenza vaccination of children and adults has
recently been highlighted [16–19], and the indirect protection of the elderly is now thought tobe more effective than direct protection. Assessing VE in children, especially in schoolchildren,

PLOS ONE | DOI:10.1371/journal.pone.0136539 August 28, 2015

2 / 15Effectiveness of Trivalent Inactivated Influenza Vaccine in Children

is essential to achieving indirect protection of the elderly, because schools are the most efficient
amplifiers of influenza epidemics in the community. The U.S. and Canada are aiming for universal immunization ranging from children 6 months of age to the elderly [20].
Influenza A(H1N1)pdm09 was the main strain in the 2013–2014 season in Japan for the
first time in 3 seasons [21]. Other epidemic viruses were influenza B and subtype A(H3N2).
The number of patients/week/sentinel (epidemic index) exceeded 1.0 at the national level (asign of the start of the epidemic season) in week 51 of 2013, and it was maintained at that levelfor 21 weeks till week 19 of 2014. The epidemic peaked in week 5 of 2014, when the incidence
was 34.4 cases/sentinel.
In the 2013–2014 season the prefectural and municipal public health institutes reported a
total 8230 isolation/detections. The influenza viruses isolated/detected in the 2013–2014 season
consisted of A(H1N1)pdm09 (43%), subtype A(H3N2) (21%), and type B (36%). A(H1N1)
pdm09 became dominant for the first time since the 2010–2011 season [21]. The ratio of
Yamagata lineage type B viruses to Victoria lineage type B viruses was 7:3.
In recent years, estimations of the effectiveness of influenza vaccine by a test-negative casecontrol design in Australia, Canada, New Zealand, the U.S., and European countries have beenreported every year [8,9,11,12,15,22–27], and this design has become the standard design forassessing VE.
It is easy to perform a very large VE study in Japan, because all children with influenza-like
illness who are seen in hospitals and clinics are tested by an influenza rapid detection test
(IRDT) [28]. In this study we used the results of the IRDT as a basis for estimating VE by atest-negative control design in children who had received TIV during the 2013–2014 season.

Methods
Study enrollment and location
Children (6 months to 15 years of age) with a fever of 38°C or over who received an IRDT in
an outpatient clinic of one of 22 hospitals in Japan (in Gunma, Tochigi, Saitama, Tokyo, Chiba,
Kanagawa, and Shizuoka prefectures) between November 9, 2013 and March 31, 2014 wereenrolled in this study.

Diagnosis of influenza
Nasopharyngeal swabs were obtained from all of the enrollees. Several IRDT different kits wereused in the hospitals, including the Espline Influenza A&B-N kit (Fujirebio Inc., Tokyo,
Japan), ImmunoAce FLU kit with LineJudge pdm kit (Tauns Laboratories, INC, Shizuoka,
Japan), Quick Chaser Flu A, B kit (Mizuho Medy Co., Ltd., Saga, Japan), QuickNavi-Flu kit(DENKA SEIKEN Co., Ltd., Tokyo, Japan), and Clearline Influenza A/B/(H1N1)2009 kit(Alere Medical Co., Ltd., Tokyo, Japan). All of these IRDT kits are capable of differentiating
between influenza A and influenza B. Four of the 22 hospitals used the Clearline Influenza A/
B/(H1N1)2009 kit, or LineJudge pdm kit, which enables differentiate between influenza A,
influenza B, and influenza A H1N1pdm09. All of the IRDT kits have similar sensitivities(88%–100%) and specificities (94%–100%) according to their manuals [29].

Case and control patient identification
All participants who visited any of 22 hospitals located in the Kanto region in Japan between
November 6 2013 and March 2014 were included in the study. Case patients were identified as
patients who were IRDT-positive, and control patients were identified as patients who were
IRDT-negative during the same period. Both the case patients’ and control patients’ medical

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3 / 15Effectiveness of Trivalent Inactivated Influenza Vaccine in Children

charts were reviewed, and information regarding symptoms, influenza vaccination, number ofvaccine doses (one or two), influenza complications and hospitalizations, gender, age, comorbidities, and treatment with neuraminidase inhibitors (NAIs) was collected and recorded. We
excluded children for whom definite information on influenza vaccination was unavailable.

VaccineA trivalent inactivated subunit-antigen vaccine is used in Japan. The vaccine strains to produce
the vaccine for use in the 2013–2014 season were A/California/7/2009(X-179A) for A(H1N1)
pdm09, A/Texas/50/2012(X-223) for H3N2, and B/Massachusetts/02/2012(BX-51B) for type
B, Yamagata lineage.
In Japan, two 0.25 ml doses of vaccine 2 to 4 weeks apart are recommended for children 6months to 2 years of age, and two 0.50 ml doses of vaccine 2 to 4 weeks apart are recommended
for children 3–12 years of age. Only one 0.5 ml dose of vaccine is recommended for children
over 13 years of age and over. The children in the vaccinated group in this study were immunized at our hospitals or elsewhere.

Test-negative case-control design
VE was estimated by a test-negative case-control design in which a patient who presented witha fever of 38°C or over and was IRDT-positive for influenza virus was considered a case, and apatient who presented with a fever of 38°C or over and was IRDT-negative was considered acontrol. VE was defined as “1- OR (odds ratio)”, and OR was calculated as(no. influenza-positive among vaccinated patients x influenza-negative amongunvaccinated)___________________________________________(no. influenza-negative among vaccinated patients x no. influenza-positive among unvaccinated patients).
We calculated VE and adjusted VE as shown in Statistical Analysis.

Statistical Analysis
The statistical analysis was performed by using the SPSS. Ver. 22 software program (IBM,USA) and “Excel Tokei (Statistics) 2012 for Windows” software program (Social Survey
Research Information Co., Ltd., Tokyo, Japan).
VE was adjusted for age group (6–11 months, 1–2 years, 3–5 years, 6–12 years and 13–15
years), comorbidity (yes or no), area of the Kanto region of Japan, i.e., (north area: Gunma Prefecture and Tochigi Prefecture; middle area: Saitama Prefecture, Tokyo Prefecture, and Chiba
Prefecture; and south area: Kanagawa Prefecture and Shizuoka Prefecture), and month of onsetof illness.
We also estimated VE according to the number of doses of vaccine administered, the phaseof the season, and area of the Kanto region where the hospitals were located, and we assessed
VE in preventing hospitalization. The Breslow Day test was used to assess the homogeneity ofthe odds ratios in several 2 × 2 contingency tables.

EthicsThis study was approved by The Keio University Ethics Committee in 2013 (No. 20130216)
and the Institutional Review Board (IRB) at each hospital. Eligible patients and their guardians(usually parents) were informed about the study objects and methods verbally at the outpatient
departments. We recorded the necessary information from patients and the guardians using a

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4 / 15Effectiveness of Trivalent Inactivated Influenza Vaccine in Children

standardized questionnaire sheets, when we obtained the consent to be enrolled to this study.
The requirement for obtaining written consent was waived by IRBs because testing patientswith IRDT is a standard practice in Japan.

Results
Characteristics of the enrollees
A total of 4970 children were enrolled in this study, but 243 were subsequently excluded fromthe analysis for the following reasons: 117 were <6 months old or >15 years old; 73 had a fever<38°C; 22 were examined twice during one episode (the data for the first visit for each patient
was deleted); 30 had an unclear influenza vaccination history; and since one was both influenza
A and B positive, there was a possibility of being false-positive. Of the remaining 4727 patients
who were eligible for the analysis in this study (Table 1). 876 had influenza A, 66 of whom wereconfirmed to have A(H1N1)pdm09 infection, the other 810 patients had influenza A subtype
unknown; and 1405 patients had influenza B. Of the 4727 subjects of the analysis, 2446 were
IRDT-negative.
Table 1 shows the characteristics of the enrollees. The comorbidities consisted of respiratory
comorbidities (n = 422), including asthma, neurological comorbidities (n = 128), including epilepsy, cardiac comorbidities (n = 57), allergic comorbidities (n = 49), renal comorbidities(n = 30), endocrinological comorbidities (n = 28), immunological comorbidities (n = 4), andother comorbidities (n = 96).

VE against influenza
Influenza vaccine was effective against influenza virus infection overall (Table 2). It was more
effective against influenza A infection than against influenza B infection (62% vs. 32%;
p < 0.001, Breslow-Day test) and very effective against A(H1N1)pdm09, against which its VE
was as high as 77% (95% confidence interval [CI]: 59 to 87).
No statistically significant VE was detected in the infant group 6 months to 11 months ofage, but the influenza vaccine was significantly more effective in the 1–12-year-old group.
Adjusted VE in the 1–2-year-old group (63%, 95% CI: 51 to 72) and 3–5-year-old group (60%,
95% CI: 49 to 69) was similar. VE was 36% (95% CI: 23 to 47) in the 6–12-year-old group andsignificantly lower than the 58% (95% CI: 48 to 67) in the 3–5-year-old group (p = 0.0049, Breslow-Day test).
By contrast, adjusted VE against influenza A(H1N1)pdm09 increased with the age group:from 67% (95% CI: 15 to 87) in the 1–2-year-old group, to 84% (95% CI: 43 to 96) in the 3–
5-year-old group, and 90% (95% CI: 67 to 97) in the 6–12-year-old group. No statistically significant VE was shown against influenza A, A(H1N1)pdm09, or influenza B in the 13–15-yearold group.
Adjusted VE in the 6 months to 23 months group was 56% (95% CI: 38–69), 62% (95% CI:
43–75) against influenza A, 60% (95% CI: -35-88) against A(H1N1)pdm09, and 38% (95% CI:-9-64) against influenza B.

Number of doses of vaccine
Two doses of influenza vaccine provided better protection against influenza A infection than
only one dose in the group of children 6 months to 12 years of age (Table 3). The OR of two
doses versus one dose was 0.72 (95% CI: 0.52 to 0.99). No significant difference in protection
between the two doses was found against influenza B infection.

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5 / 15Effectiveness of Trivalent Inactivated Influenza Vaccine in Children

Table 1. Characteristics of Enrollees in 2013/14 Influenza Season.

Sex

Age

Comorbidity

Province

Months of Onset

Visit (hours after onset)

Received Vaccine in 2013/2014

Vaccine Dose in 2013/2014

Treatment with NAIs*

Any Inﬂuenza (%)

Type A (%)

H1N1pdm09a (%)aType B (%)

Inﬂuenza Negative (%)

female1082 (47)404 (46)

26 (39)678 (48)1096 (45)male1199 (53)472 (54)40 (61)727 (52)

1349 (55)

total

2281

876

66

1405

2445

6-11m/o49 (2)

39 (4)

2 (3)

10 (1)

166 (7)

1–2 y/o342 (15)224 (26)

21 (32)118 (8)803 (33)

3–5 y/o539 (24)248 (28)

17 (26)291 (21)675 (28)

6–12 y/o1169 (51)331 (38)

23 (35)838 (60)694 (28)

13–15 y/o

182 (8)34 (4)3 (5)148 (11)108 (4)

total

2281

876

66

1405

2446

No

1802 (82)692 (83)

39 (64)1110 (81)1874 (82)Yes

401 (18)146 (17)22 (36)255 (19)413 (18)

total

2203

838

61

1365

2287
261 (11)north

264 (12)125 (14)36 (55)139 (10)

middle1226 (54)496 (57)

30 (45)730 (52)

1356 (55)south

791 (35)

255 (29)0536 (38)829 (34)

total

2281

876

66

1405

2446

Nov 2013

0 (0)

0 (0)

0 (0)

0 (0)

12 (0)

Dec 2013

66 (3)

25 (3)1 (2)

41 (3)

272 (11)

Jan 2014

609 (27)424 (48)

32 (48)185 (13)629 (26)

Feb 2014

1017 (45)345 (39)

27 (41)672 (48)838 (34)

Mar 2014

589 (26)

82 (9)6 (9)507 (36)695 (28)

total

2281

876

66

1405

2446

<12 h

695(32)287 (35)

23 (35)408 (30)757 (33)

12–48 h1312 (60)498 (60)

40 (61)814 (59)1266 (55)>48 h

187 (9)39 (5)3 (5)148 (11)281 (12)

total

2194

824

66

1370

2304

>12 h

1499

537

43

962

1547

No

1401 (61)661 (70)

49 (74)790 (56)1143 (47)Yes

880 (39)265 (30)

17 (26)615 (44)1303 (52)

total

2281

876

66

1405

2446

none

1401 (62)611 (70)

49 (74)790 (56)1143 (47)once

219 (10)71 (8)5 (8)148 (11)270 (11)twice

653 (29)

192 (22)12 (18)461 (33)

1010 (42)

total

2273

874

66

1399

2423

No

72 (5)

22 (4)

050 (5)

1485 (96)Any1527 (95)604 (96)35 (100)923 (95)

60 (4)

total

1599

626

35

973

1545

Difference between Any Inﬂuenza and Inﬂuenza Negative

P = 0.07b

P<0.001c

P = 0.90b

p = 0.43 dp<0.001 e

p = 0.398 f

p<0.001 b

p<0.001 g

p<0.001 h

a Only four hospitals used IRDTs that can detect H1N1pdm09.
b Chi-square test.
c Mann–Whitney U- test.
d Chi-square test, Cramer's V = 0.0189.
e Chi-square test, Cramer's V = 0.1830.
f Chi-square test, comparing the number of patients who visited <12 hours after the onset with the number who visited later.
g number who visited later.
h Chi-square test, Cramer's V = 0.9160.
* Neuraminidase Inhibitors.
doi:10.1371/journal.pone.0136539.t001

Protection against hospitalization
Influenza vaccination was effective in preventing hospitalization (Table 4), especially for influenza A virus infection (76%, 95% CI: 51 to 88). VE was as high as 90% (95% CI: 54 to 98) in

PLOS ONE | DOI:10.1371/journal.pone.0136539 August 28, 2015

6 / 15PLOS ONE | DOI:10.1371/journal.pone.0136539 August 28, 2015

Adjusted a,b,d

29 (-17-57)
22 (-33-54)

Adjusted a

39 (26–50)

Crude

36 (23–47)

Crude

60 (49–69)

Adjusted a

58 (48–67)

Crude

57 (44–67)

Adjusted a

55 (42–65)

Crude

Adjusted a

61 (49–70)
63 (51–72)

Crude

21 (-87-67)

Adjusted a

Adjusteda51 (43–58)

Adjusted a,b,c
24 (-77-68)

45 (38–52)

Adjusted a,b

Crude

45 (38–51)
45 (39–52)

Crude(55/182) [41/108](495/1169) [371/694](208/539) [406/675](122/391) [485/969](114/342) [451/803](8/49) [34/166](880/2281) [1303/2446]*

12 (-115-64)

32 (-57-70)

58 (44–69)

55 (41–66)

73 (63–81)

72 (62–80)

67 (54–76)

65 (52–74)

72 (60–80)

70 (58–78)

30 (-85-74)

29 (-82-73)

67(59–74)

63 (56–69)

63 (56–69)

62 (55–68)

VE%(95% CI)

VE%(95% CI)(10/34) [41/108](113/331) [371/694](73/248) [406/675](69/263) [485/969](63/224) [451/803](6/39) [34/166](265/876) [1303/2446]*

doi:10.1371/journal.pone.0136539.t002* (vaccinated/cases) [vaccinated/controls].

30 (-2-52)

23 (-34-56)

29 (-21-58)

27 (11–40)

44 (25–58)

43 (24–57)

29 (-6-52)

NA g
NA g

e Two hospitals have no information on comorbidity.
f Only four institutes used IRDTs that can detect A(H1N1)pdm09. One hospital had no information on comorbidity.

g Not analyzed because few patients developed inﬂuenza.

41(12–60)
41 (10–61)

30 (13–43)(1/3) [8/18](5/23) [85/123](3/17) [78/134](8/23) [110/193](8/21) [102/157]

NA g

90 (67–97)

88 (64–96)

84 (43–96)

85 (44–96)

62 (4–85)

60 (1–84)

67 (15–87)

67 (15–87)

NA

g

NA g

25 (13–35)
33 (20–45)

Data for 3046 patients were available; 1499 for any inﬂuenza (A; 537, H1N1; 43, B; 962) and 1547 for inﬂuenza negative.

c Adjusted for time tested after the onset (<12, 12–48 and >48 hours).
d Patients tested only >12 hours after onset.

b Adjusted for age (0–15 y/o).

32 (22–40)
26 (14–36)

NA g(0/2) [8/36](17/66) [281/468]*

VE%(95% CI)

Type B e

85 (65–93)

77 (59–88)

77 (59–87)

77 (59–87)

VE%(95% CI)

A(H1N1)pdm09 f

a Adjusted for comorbidity (yes or no, except H1N1 analysis), area (north area, middle area, south of the Kanto region), months of onset.

Age 13–15 y/oAge 6–12 y/o

Age 3–5 y/oAge 6 m/o-2 y/o

Age 1–2 y/o

Age 6–11 m/o

All Ages 6 mo-15 y/oType Ae

Any Inﬂuenzae

Table 2. Effectiveness of Influenza Vaccine for Children in 2013/14 Influenza Season (N = 4727).

(45/148) [41/108](382/838) [371/694](135/291) [406/675](53/128) [485/969](51/118) [451/803](2/10) [34/166](615/1405) [1303/2446]*

Effectiveness of Trivalent Inactivated Influenza Vaccine in Children

7 / 15Effectiveness of Trivalent Inactivated Influenza Vaccine in Children

Table 3. Effectiveness of Influenza Vaccine for Children, by Vaccine Doses (6m/o-12y/o).

Any Inﬂuenza

Type A

A(H1N1)pdma

Type B

Vaccine Doses

Cases

Controls

OR

95% CI
0.50–0.76

none

1274

1076

0.62 (non vs once)

once

172

235

0.54 (non vs twice)

0.48–0.62

twice

646

1004

0.88 (once vs twice)

0.71–1.10

none

587

1076

0.48 (non vs once)

0.36–0.65

once

62

235

0.35 (non vs twice)

0.29–0.42

twice

191

1004

0.72 (once vs twice)

0.52–0.99

none

47

177

0.38 (non vs once)

0.38–1.11

once

4

40

0.20 (non vs twice)

0.10–0.38

twice

12

228

0.53 (once vs twice)

0.16–1.71

none

687

1076

0.73 (non vs once)

0.57–0.94

once

110

235

0.71 (non vs twice)

0.61–0.82

twice

455

1004

0.97 (once vs twice)

0.75–1.25

a Among the 4 hospitals which used IRDTs that can detect H1N1pdm09.
doi:10.1371/journal.pone.0136539.t003

preventing hospitalization for influenza A(H1N1)pdm09 infection. By contrast, the TIV wasnot effective in preventing hospitalization for influenza B virus infection.
The reason for hospitalization was known in 14 (15%) of the 92 hospitalizations, and it was
pneumonia in 5 cases, encephalopathy in 4 cases, asthma in 2 cases, mild consciousness disturbance in 1 case, seizure clusters in 1 case, and co-infection with Mycoplasma pneumoniae in 1 case.

VE by month of onset of illness
VE was lower in the late phase of the influenza epidemic (Table 5), decreasing from 59% (95%
CI: 49 to 66) in the 3-month period November, December, and January to 39% (95% CI: 29 to47) in the 2-month period February and March. VE against influenza B was only 31% (95% CI:
19 to 40) in the 2-month period February and March. The number of influenza B patients wasmuch higher in February and March, i.e., in the late phase of the epidemic.

VE according to area and hospital
There were no significant differences between VE against influenza A according to area, but
VE against influenza B was significantly higher in the north area than in the middle area orTable 4. Effectiveness of Influenza Vaccine for Preventing Influenza Hospitalization.

Any
Inﬂuenza

Immunization
StatusNo
Hospitalization

Hospitalization with
Inﬂuenza

unvaccinated

2080

65

vaccinated

1778

27

Type Aunvaccinated

2080

44

vaccinated

1778

9

H1N1pdm09 a

unvaccinated

223

15

vaccinated2842

unvaccinated

2080

21

vaccinated

1778

18

Type B

Effectivenes for Preventing Inﬂuenza
Hospitalization

95%
CI

51

24–69

76

51–88

90

54–98

0

-89-47a Among the 4 hospitals which used IRDTs that can detect A(H1N1)pdm09.
doi:10.1371/journal.pone.0136539.t004

PLOS ONE | DOI:10.1371/journal.pone.0136539 August 28, 20158 / 15Effectiveness of Trivalent Inactivated Influenza Vaccine in Children

Table 5. Effectiveness of Influenza Vaccine, by Phase.
Any Inﬂuenza

Type A

A(H1N1)pdm09a

Type B

VE% (95%CI)

VE% (95%CI)

VE% (95%CI)

VE% (95%
CI)

Nov, 2013-Jan,
2014

59 (49–66)(211/675) [478/913]*

66 (56–73)(123/449) [478/913]*

78 (49–91)(9/33) [82/130]*

42 (22–57)(88/226) [478/913]*

Feb-March, 201439 (29–47)(669/1606) [825/1533]

57 (46–66)(142/427) [825/1533]

78 (49–90)(8/33) [199/338]

31 (19–40)(527/1179) [825/
1533]

Total

45 (38–51)(880/2281) [1303/
2446]

62 (55–68)(265/876) [1303/
2446]

77 (59–87)(17/66) [281/468]

32 (22–40)(615/1405) [1303/
2446]

a Among four hospitals which used IRDTs that can detect A(H1N1)pdm09.
* (vaccinated/cases) [vaccinated/controls].
doi:10.1371/journal.pone.0136539.t005

south area (55% in the north area vs. 27–28% (both p < 0.05) (Table 6). The VE values according to hospital ranged widely from -275% to 84% against influenza B. By contrast, VE values
against influenza A were similar in most of the hospitals.

Vaccine coverage
The proportion of vaccine coverage calculated for the IRDT-negative enrollees was 53% (1303/
2446). By age group, it was: 6–11 months, 21% (34/166); 1–5 years, 58% (857/1478); for 6–12
years, 53% (371/694); and 13–15 years, 38% (41/108).

Discussion
In this large study of over 4700 children 6 months to 15 years of age the overall influenza VE
for prevention of laboratory-confirmed medically attended influenza illness was 46% (Table 2).
High VE was shown in the influenza A group (63%), and VE was as high as 77% in the groupwith confirmed A(H1N1)pdm09 infection. Our results were consistent with reports in othercountries where A(H1N1)pdm09 was the main epidemic virus [12,13]. In the 2013–2014 season in Japan, both A(H3N2) and A(H1N1)pdm09 were co-circulating, but A(H1N1)pdm09
was the dominant strain [21].
VE against influenza A was over 72% in the group of children 1–5 years of age (72% in the
1–2-year-old group, 73% in the 3–5-year-old group) and was higher than VE in the 6–12-yearold group (58%) (Table 2). By contrast, VE against A(H1N1)pdm09 increased with the agegroups, from 67% in the 1–2-year-old group, to 84% in the 3–5-year-old group, and 90% in the
6–12-year-old group. Most of the children in the 6–15-year-old group had been infected in theA(H1N1)pdm09 pandemic in 2009–2010 [30] and probably had sufficient residual immunity
against A(H1N1)pdm09 from five years before. The pre-epidemic measurement of hemagglutination inhibition (HI) titers showed that 60%–70% of the children 5–15 years of age in Japan
had HI titers to A(H1N1)pdm09 that were over 1:40 [31]. Because no antigenic changes in A
(H1N1)pdm09 have been reported, the majority of children over 6 years of age who were
IRDT-positive for influenza A probably had an A(H3N2) infection, and that would have led to
the lower effectiveness against influenza A in this age group. In other studies VE has been estimated to be low in patients infected with A/H3N2 [8,9]. VE has not been demonstrated in children 13–15 years of age, because the number of children tested was small and/or they had
mainly contracted influenza A/H3N2.
VE against influenza B in the present study was low, only 26% (Table 2). Lower vaccine efficacy against influenza B than against influenza A has been postulated in Japan, especially in

PLOS ONE | DOI:10.1371/journal.pone.0136539 August 28, 2015

9 / 15Effectiveness of Trivalent Inactivated Influenza Vaccine in Children

Table 6. Effectiveness of Influenza Vaccine in Children (6 m/o-12 y/o) According to Hospital (A to V).

Area
North

vaccineb

vaccineb

noc

A(H1N1)
pdm09 aType B

noc

vaccineb

noc

7

29

41

68

15

36

B

20

47

7

22

C

28

60

14

31

89

175

36

89

7

29

D

20

23

4

13

0

4

E

49

105

14

F

14

33

G

99

H

vaccineb

Inﬂuenza
Negativenoc

vaccineb

VE (95%CI)
noc

Any
InﬂuenzaType A

A(H1N1)pdm09 a
84 (62–93)

Type Bd

26

32

116

77

60 (35–75)

72 (46–86)

13

25

4

3

68 (-56-93)

76 (-33-96)

14

29

31

30

55 (11–77)

56 (2–80)

53

86

151

110

63 (47–74)

71 (53–81)

82 (62–93)

55 (32–71)

16

10

58

54

19 (-64-60)

71 (7–91)

100-49 (-257-38)

63

35

42

99

92

57 (33–72)

79 (61–89)

23 (-32-54)

4

17

10

16

37

36

59 (10–81)

77 (25–93)

39 (-52-76)

152

46

71

53

81

126

90

53 (33–68)

54 (27–71)

79

55

20

24

6

10

59

31

65

34

25 (-29-56)

56 (10–79)

69 (6–89)

0 (-82-45)

I

23

20

12

11

4

6

11

9

42

22

40 (-33-73)

43 (-50-78)

65 (-37-91)

36 (-78-77)

J

78

67

19

23

59

44

79

41

40 (0–63)

57 (12–79)

K

55

95

19

43

36

52

102

92

48 (19–66)

60 (27–78)

38 (-4-62)

L

14

23

10

16

4

7

41

44

35 (-44-70)

33 (-65-73)

39 (-125-83)

M

82

140

17

50

513

713

165

331

N

78

194

21

O

73

141

11

P

8

16

Q

7

R
S

46 (2–70)
61 (-101-92)
53 (-5-79)

53 (27–70)

30 (-20-60)

65

90

106

96

47 (22–64)

69 (43–83)

348

382

755

601

43 (33–51)

60 (51–68)

85

57

109

111

150

46 (22–62)

67 (43–80)

29 (-6-53)

25

62

116

90

123

29 (-5-52)

40 (-29-72)

27 (-10-52)

2

7

6

9

41

61

26 (-90-71)

57 (-11592)

1 (-200-67)

7

2

5

5

2

6

9

-50 (-553-66)

40 (-31791)-275 (-250446)

45

40

9

25

36

15

68

29

52 (12–74)

85 (63–94)-2 (-115-51)

42

74

10

24

32

50

36

34

46 (2–71)

61 (6–84)

40 (-15-68)

T

2

6

0

1

2

5

10

4

87 (4–98)

100

84 (-19-98)

U

14

23

7

14

7

9

17

15

46 (-40-79)

56 (-38-86)

31 (-130-79)
61 (-52-90)

MiddleTotal
SouthType A

A

North Total
MiddleAny Inﬂuenza

V

10

20

9

12

2

5

7

7

18

7

71 (0–91)

84 (0–98)

South Total

278

513

64

191

0

0

214

322

397

432

41 (28–52)

64 (50–73)

Total

880

1401

265

611

17

49

615

790

1303

1143

45 (38–51)

62 (55–68)

35 (0–57)
67 (26–85)

27 (13–39)

28 (10–42)
77 (59–87)

32 (22–40)

a Among four hospitals which used IRDTs that can detect A(H1N1)pdm09.
b Number of patients vaccinated.
c Number of patients unvaccinated.
d VE against any inﬂuenza and VE against inﬂuenza B were higher in the north area than in the middle or south area (Breslow-Day, p < 0.05).
doi:10.1371/journal.pone.0136539.t006

young children [4]. Low VE against influenza B probably reflects the characteristic epidemic
pattern of influenza B in Japan: influenza B virus epidemics usually start in February, after theend of the influenza A epidemic, and continue through March to April. Since children in Japan
usually receive influenza vaccine in October and November, the level of antibody titer generated in response to the vaccine may be lower in March when influenza B epidemics peak. Ourdata showed that VE against influenza A and B was lower in February and March (Table 5).
The lower VE against influenza B was probably attributable to waning immunity in response to
the vaccine, as has been reported in regard to the effectiveness of vaccines against influenza A
H3N2 [26,32], and the low VE against influenza B was partly attributable to the mixed epidemic of influenza B lineages, i.e., of B/Victoria and B/Yamagata, in the 2013–2014 season in
Japan [21], although B/Yamagata was both the main epidemic strain and the vaccine strain. Blineage mismatches present a greater obstacle to vaccine efficacy in children [3]. In the near
future, quadrivalent vaccines that contain both B-lineage antigens will be introduced in Japan,

PLOS ONE | DOI:10.1371/journal.pone.0136539 August 28, 201510 / 15Effectiveness of Trivalent Inactivated Influenza Vaccine in Children

thereby reducing B-lineage mismatches. Low HI titer responses to influenza B strain are of particular concern in children who receive influenza vaccine [3], because influenza B infection iscommon in children and as serious clinically as influenza A infection. NAIs are also known tobe less effective against influenza B [33].
In this study influenza vaccine was not effective against either influenza A (30%, 95% CI-85, 74) or influenza B (-45%, 95% CI -684, 75) in 6- to 11-month-old infants, but it was effective in children over 1 year old (Table 2). Indirect protection attributable to the mass vaccination program of schoolchildren in Japan in the 1960s to 1980s protected young children 1–4
years of age against influenza encephalopathy [17,19]. Young children did not receive influenza
vaccine in the 1960s to 1980s. On the other hand, traditional cohort studies to estimate VE
may have yielded excessively high VE for infants. One study reported 42%-69% VE in infants[34]. Since siblings and/or parents in families in which an infant has received influenza vaccine
have usually also received influenza vaccine, thereby providing highly effective indirect protection to their infants whether indirect protection exists in the background should be notedwhen interpreting reports on VE in children.
Two doses of influenza vaccine have been reported to be necessary to provide sufficient protection in children, especially in young children [6,7,35,36]. However, the results of the present
study showed that only one dose of influenza vaccine was effective in protecting against influenza A (52%) and influenza B (27%). Two doses of influenza vaccine were needed to optimize
protection only against influenza A in the present study (one dose vs. two doses, OR = 0.72)(Table 3). However, a second dose did not have any additive effect against influenza B.
A meta-analysis of VE in children showed no convincing evidence that influenza vaccine
can reduce mortality, hospitalizations, or serious complications [37]. However, the results ofthe present study demonstrated that influenza vaccination was effective in reducing hospitalization of children infected with influenza A infection by as much as 76% and of children
infected with A(H1N1)pdm09 infection by as much as 90% (Table 4). On the other hand, noeffectiveness in preventing hospitalization for influenza B was shown. By contrast, in the 2002–
2008 seasons VE for preventing hospitalization with influenza A and B in children 6 months to
5 years old was reported to be 71% and 72% [34]. There is a recent report of a study showingthat influenza vaccination was associated with a three-quarters reduction in the risk of lifethreatening influenza illness in children 6 months to 17 years of age [38]. Moreover, VE against
hospitalization with laboratory-confirmed influenza A and B has been estimated to be 61.7%[39].
The results of this study showed clear differences between VE against influenza B according
to area (Table 6). In the 2013–2014 season, the ratio of Yamagata lineage viruses to Victoria
lineage viruses was 7:3 nationwide [21]. Since the vaccine virus was the Yamagata lineage inthe 2013–2014 season, Yamagata lineage viruses may have been more epidemic in the northarea than in the other areas of the Kanto region.
The limitations of this study need to be considered. Unlike most previous test-negative
case-control design studies based on PCR data, this study was based on the results of IRDTs.
Suzuki et al. [40] found no difference between VE estimated on the basis of IRDT results on
the basis of PCR data. However, the use of IRDTs in test-negative studies may result in underestimations of VE. Orenstein et al. [41] reported that the bias toward underestimating true VE
introduced by low test specificity increases and lesser degree, by low test sensitivity. In a casecontrol study on influenza VE based IRDT results [5], it was postulated that virus shedding isgreatest during the first days of influenza infection and therefore, patients who were tested during the early phase of the illness were more likely to test positive. In our study, about 90% of
the enrollees received IRDT within 48 hours of onset of illness (Table 1).

PLOS ONE | DOI:10.1371/journal.pone.0136539 August 28, 201511 / 15Effectiveness of Trivalent Inactivated Influenza Vaccine in Children

On the other hand, the sensitivity of IRDTs is low when patients are tested within 12 hours
after the onset of influenza illness because of the low virus infection titers in the upper respiratory tract in the early phase of infection [42]. We therefore selected patients who were tested 12hours or more after the onset of influenza illness, and when we estimated the VE of the influenza vaccine in that group of patients, we found that it was slightly higher (+4% to +8%)
(Table 2). When estimating VE by a test-negative case-control design based on IRDT results, it
is better to select patients tested 12 hours or more after the onset of influenza-like illness.
Almost all children with influenza-like illness in Japan receive an IRDT, and if positive, they
are treated with NAIs [28]. This diagnosis and treatment system established in Japan worked
well during the pandemic caused by A(H1N1)pdm09 and resulted in the very low fatality ratein Japan during that pandemic [30]. In future epidemics in Japan, VE estimated by a test-negative case-control design based on IRDT results from various parts of Japan will be reported rapidly. The large number of cases in Japan makes it possible to estimate VE with considerable
precision, and the most appropriate vaccination policy should be established based on the dataobtained.

Acknowledgments
This study would not have been possible without the excellent support from all the doctors of
the Department of Pediatrics, Keio University School of Medicine.
Members of the Keio Pediatric Influenza Research Group. Coordination: ^T Takahashi,
Department of Pediatrics, Keio University School of Medicine in Tokyo; ^N Sugaya, Department of Paediatrics, Keiyu Hospital in Yokohama. Data management: ^M Shinjoh, Department of Pediatrics, Keio University School of Medicine in Tokyo; ^S Sekiguchi, Department ofPediatrics, Keio University School of Medicine in Tokyo. Epidemiology and Methodology:^C Kawakami, Yokohama City Institute of Health in Yokohama. Data collection (Pediatricians): ^Y Yamaguchi, Department of Clinical Research, National Hospital Organization,
Tochigi Medical Center in Utsunomiya; ^Y Tomidokoro, Department of Pediatrics, Tokyo
Metropolitan Ohtsuka Hospital in Tokyo; ^M Fujino, Department of Pediatrics, Saiseikai Central Hospital in Tokyo; ^H Shiro, Department of Pediatrics, Yokohama Rosai Hospital in
Yokohama; ^O Komiyama, Department of Pediatrics, National Hospital Organization, Tokyo
Medical Center in Tokyo; ^N Taguchi, Department of Pediatrics, Keiyu Hospital in Yokohama; ^Y Nakata, Department of Pediatrics, Nippon Kokan Hospital in Kawasaki; ^N
Yoshida, Department of Pediatrics, Kyosai Tachikawa Hospital in Tachikawa; ^A Narabayashi,
Department of Paediatrics, Kawasaki Municipal Hospital in Kawasaki; ^M Myokai, Department of Pediatrics, Shizuoka City Shimizu Hospital in Shizuoka; ^M Sato, Department of Pediatrics, Tokyo Dental College Ichikawa General Hospital in Ichikawa; ^M Furuichi,
Department of Pediatrics, Saitama City Hospital in Saitama; ^H Baba, Department of Pediatrics, Fuji Heavy Industries Health Insurance Society Ota Memorial Hospital in Ota; ^H Fujita,
Department of Pediatrics, Hiratsuka Kyosai Hospital in Hiratsuka; ^A Sato, Department ofPediatrics, Yokohama Municipal Citizen's hospital in Yokohama; ^I Ookawara, Department ofPediatrics, Japanese Red Cross Shizuoka Hospital in Shizuoka; ^K Tsunematsu, Department ofPediatrics, Hino Municipal Hospital in Hino; ^M Yoshida, Department of Pediatrics, Sano
Kousei General Hospital in Sano; ^M Kono, Department of Pediatrics, National Hospital
Organization Saitama National Hospital in Wako; ^F Tanaka, Department of Pediatrics, Hiratsuka City Hospital in Hiratsuka; ^T Kimiya, Department of Pediatrics, Tokyo Metropolitan
Ohtsuka Hospital in Tokyo. Steering committee: ^T Takahashi, Department of Pediatrics,
Keio University School of Medicine in Tokyo; ^S Iwata, Department of Infectious Diseases,
Keio University School of Medicine in Tokyo; M Bamba, Department of Paediatrics, Kawasaki

PLOS ONE | DOI:10.1371/journal.pone.0136539 August 28, 201512 / 15Effectiveness of Trivalent Inactivated Influenza Vaccine in Children

Municipal Hospital in Kawasaki; ^K Mitamura, Department of Pediatrics, Eiju General Hospital in Tokyo; S Sato, Department of Pediatrics, Saitama City Hospital in Saitama; Y Yamashita,
Department of Pediatrics, Yokohama Municipal Citizen's hospital in Yokohama; M Anzo,
Department of Paediatrics, Kawasaki Municipal Hospital in Kawasaki; ^M Shinjoh, Department of Pediatrics, Keio University School of Medicine in Tokyo; ^N Sugaya, Department ofPaediatrics, Keiyu Hospital in Yokohama.

Author Contributions
Conceived and designed the experiments: MS NS TT SI. Performed the experiments: YY YT SS
KM M. Fujino HS OK NT YN NY AN MM MS M. Furuichi HB HF AS IO KT MY MK FT TK.
Analyzed the data: MS NS YY YT TT SI. Contributed reagents/materials/analysis tools: NS CK.
Wrote the paper: MS NS.

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