Influenza virus seroincidence in a cohort of healthy and high-risk children enrolled in infancy, Bangkok, Thailand

Abstract

Background:
We measured seroconversion to influenza viruses and incidence of symptomatic influenza virus infection in a cohort of children in Bangkok, Thailand.

Methods:
Children aged ≤6 months were followed for two years for acute respiratory illness (ARI) and had serum specimens taken at 6-month intervals and tested by hemagglutination inhibition (HI) assay. Seroconversion was defined as a >4-fold rise in the HI titers between time points with a titer of >40 in the second specimen. Respiratory swabs were tested by rRT-PCR for influenza. Data were analyzed using generalized linear models.

Results:
Of 350 children, 266 (76%, 147 were healthy and 119 were high-risk) had ≥2 serum specimens collected before influenza vaccination. During the 2-year follow-up, 266 children contributed 370 person-years of observation, excluding post-vaccination periods. We identified 32 ARI cases with rRT-PCR-confirmed influenza virus infection (7 infections/100 person-years, 95% confidence interval [CI], 4-11). There were 126 episodes of influenza virus infection, resulting in a seroconversion rate of 35 infections/100 person-years (95% CI, 30-42). Rates in healthy and high-risk children did not differ.

Conclusions:
Influenza virus infection is common during the first two years of life among Thai children. A large proportion of infections may not be detected using the ARI case definition.

Full text

Influenza
virus
seroincidence
in
a
cohort
of
healthy
and
high-risk
children
enrolled
in
infancy,
Bangkok,
Thailand
Kamonthip
Rungrojcharoenkit
a,1
,
Wanitchaya
Kittikraisak
b,1,
*,
Darunee
Ditsungnoen
b
,
Sonja
J.
Olsen
c
,
Piyarat
Suntarattiwong
d
,
Tawee
Chotpitayasunondh
d
,
Chonticha
Klungthong
a
,
In-Kyu
Yoon
a
,
Fatimah
S.
Dawood
c
,
Stefan
Fernandez
a
,
Louis
Macareo
a
,
Kim
A.
Lindblade
b,c
a
Virolgy
Department,
Armed
Forces
Research
Institute
of
Medical
Sciences,
Bangkok,
Thailand
b
Influenza
Program,
Thailand
Ministry
of
Public
Health
-
U.S.
Centers
for
Disease
Control
and
Prevention
Collaboration,
Nonthaburi,
Thailand
c
Influenza
Division,
U.S.
Centers
for
Disease
Control
and
Prevention,
Georgia,
USA
d
Queen
Sirikit
National
Institute
of
Child
Health,
Bangkok,
Thailand
A
R
T
I
C
L
E
I
N
F
O
Article
history:
Received
15
July
2019
Received
in
revised
form
16
August
2019
Accepted
21
August
2019
Corresponding
Editor:
Eskild
Petersen,
Aar-
hus,
Denmark
Keywords:
Seroconversion
Seroincidence
Influenza
Pediatric
Thailand
A
B
S
T
R
A
C
T
Background:
We
measured
seroconversion
to
influenza
viruses
and
incidence
of
symptomatic
influenza
virus
infection
in
a
cohort
of
children
in
Bangkok,
Thailand.
Methods:
Children
aged
6
months
were
followed
for
two
years
for
acute
respiratory
illness
(ARI)
and
had
serum
specimens
taken
at
6-month
intervals
and
tested
by
hemagglutination
inhibition
(HI)
assay.
Seroconversion
was
defined
as
a
>4-fold
rise
in
the
HI
titers
between
time
points
with
a
titer
of
>40
in
the
second
specimen.
Respiratory
swabs
were
tested
by
rRT-PCR
for
influenza.
Data
were
analyzed
using
generalized
linear
models.
Results:
Of
350
children,
266
(76%,
147
were
healthy
and
119
were
high-risk)
had
2
serum
specimens
collected
before
influenza
vaccination.
During
the
2-year
follow-up,
266
children
contributed
370
person-years
of
observation,
excluding
post-vaccination
periods.
We
identified
32
ARI
cases
with
rRT-PCR-confirmed
influenza
virus
infection
(7
infections/100
person-years,
95%
confidence
interval
[CI],
4–11).
There
were
126
episodes
of
influenza
virus
infection,
resulting
in
a
seroconversion
rate
of
35
infections/100
person-years
(95%
CI,
30–42).
Rates
in
healthy
and
high-risk
children
did
not
differ.
Conclusions:
Influenza
virus
infection
is
common
during
the
first
two
years
of
life
among
Thai
children.
A
large
proportion
of
infections
may
not
be
detected
using
the
ARI
case
definition.
Published
by
Elsevier
Ltd
on
behalf
of
International
Society
for
Infectious
Diseases.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Background
Annual
influenza
epidemics
cause
between
3–5
million
cases
of
severe
illness
and
approximately
290,000
to
600,000
deaths
globally
(World
Health
Organization,
2018).
Young
children
experience
a
high
burden
of
influenza-associated
illness,
including
hospital
admissions
for
acute
lower
respiratory
infection
(ALRI)
and
pneumonia
(Thompson
et
al.,
2004;
Neuzil
et
al.,
2002;
Poehling
et
al.,
2006;
Nair
et
al.,
2011).
Influenza
virus
infections
also
account
for
a
substantial
proportion
of
all
pneumonia
deaths
of
viral
etiology
in
young
children
globally
(Rudan
et
al.,
2008),
with
up
to
111,500
global
deaths
attributed
to
influenza-associated
ALRI
in
children
aged
<5
years
in
2008
(Nair
et
al.,
2011)
and
an
average
of
9,000–106,000
deaths
annually
among
children
aged
<5
years
in
92
countries
with
high
respiratory
infection
mortality
rates
(Iuliano
et
al.,
2018).
In
Thailand,
children
aged
<5
years
are
more
likely
to
be
hospitalized
with
influenza-associated
pneumonia
compared
to
the
general
Thai
population
(Katz
et
al.,
2007).
Estimated
annual
incidence
of
laboratory
confirmed-influenza
virus
infection
ranged
from
<100
cases
per
100,000
children
in
2003-2004
to
>400
cases
per
100,000
children
in
the
pandemic
year
(2009)
(Katz
et
al.,
2007;
Olsen
et
al.,
2010;
Baggett
et
al.,
2012).
Published
studies
of
influenza
disease
burden
in
Thailand
have
focused
on
medically
attended
illness,
and
have
used
case
definitions
that
may
not
capture
milder
or
atypical
infections,
particularly
among
young
children.
It
may
be
helpful
to
quantify
asymptomatic
infection
although
its
role
in
transmission
is
unclear.
Studies
of
persons
*
Corresponding
author
at:
Thailand
MOPH
-
U.S.
CDC
Collaboration,
Ministry
of
Public
Health,
DDC
Building
7,
Tiwanon
Road,
Nonthaburi
11000,
Thailand.
E-mail
address:
glr9@cdc.gov
(W.
Kittikraisak).
1
Both
first
authors
contributed
equally
to
this
work.
https://doi.org/10.1016/j.ijid.2019.08.022
1201-9712/Published
by
Elsevier
Ltd
on
behalf
of
International
Society
for
Infectious
Diseases.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://
creativecommons.org/licenses/by-nc-nd/4.0/).
International
Journal
of
Infectious
Diseases
89
(2019)
21–26
Contents
lists
available
at
ScienceDirect
International
Journal
of
Infectious
Diseases
journal
home
page:
www.elsevier.com/locat
e/ijid

exposed
to
influenza
who
may
or
may
not
get
ill
using
influenza
serologic
testing
to
identify
infection
based
on
changes
in
antibody
titers
over
time
have
demonstrated
that
influenza
serology
may
identify
more
infections
than
influenza
reverse
transcription
polymerase
chain
reaction
(RT-PCR)
testing
alone
(Leung
et
al.,
2015;
Monto
and
Sullivan,
1993).
However,
studies
with
longitu-
dinal
serology
testing
for
influenza
virus
infection
among
young
children
are
scarce
(Leung
et
al.,
2015).
In
this
study,
we
measured
seroconversion
to
influenza
at
six-month
intervals
over
two
years
in
a
cohort
of
children,
enrolled
during
the
first
six
months
of
life,
in
Bangkok,
Thailand,
to
calculate
subtype-specific
cumulative
incidence
of
infection
by
age
and
compare
incidence
of
influenza
virus
infection
by
health
status.
This
study
was
part
of
a
larger
cohort
study
of
respiratory
infection
in
the
pediatric
population
in
Bangkok,
Thailand
(Kittikraisak
et
al.,
2018).
Methods
Ethical
approvals
The
study
was
approved
by
the
ethics
committees
of
the
Queen
Sirikit
National
Institute
of
Child
Health
(QSNICH;
Bangkok,
Thailand)
and
the
Walter
Reed
Army
Institute
of
Research
(Maryland,
USA),
with
the
U.S.
Centers
for
Disease
Control
and
Prevention’s
Institutional
Review
Board
(Georgia,
USA)
relied
on
the
QSNICH’s
determination.
Written
parental
informed
consent
was
sought
for
all
children.
Study
population
The
study
population
consisted
of
children
receiving
care
at
the
QSNICH,
the
largest
children’s
tertiary
care
public
hospital
in
Thailand
serving
exclusively
pediatric
population
from
birth
to
18
years
of
age.
All
study
participants
were
residents
of
the
metropolitan
area
of
Bangkok
and
its
vicinity.
Enrollment
method
and
study
procedures
Enrollment
method
and
study
procedures
have
been
previously
described
(Kittikraisak
et
al.,
2018;
Kittikraisak
et
al.,
2017).
Briefly,
a
prospective
cohort
study
(Pediatric
Respiratory
Infection
Cohort
Evaluation,
the
PRICE
study)
to
determine
influenza
burden
among
children
living
in
the
Bangkok
Metropolitan
areas
who
sought
care
at
QSNICH
(for
any
cause
except
acute
respiratory
illness)
was
established
in
August
2011.
Healthy
children
aged
0–36
months
were
matched
by
age
(1
month
for
children
aged
<1
year
and
1
year
for
those
aged
1
year)
and
calendar
time
with
children
at
high-risk
for
severe
influenza
complication
(hereafter
referred
to
as
high-risk
children),
defined
as
any
child
with
one
or
more
of
the
underlying
medical
conditions
as
described
in
Supplementary
Table
S1.
None
of
the
children
were
immunocompromised.
At
enrollment,
study
nurses
administered
structured
questionnaires
to
collect
demographic
and
health
history
data.
Within
two
months
of
enrollment,
study
nurses
visited
children’s
homes
to
record
family
and
household
characteristics,
and
childhood
immunizations
from
children’s
vaccine
books
(including
influenza
vaccination
if
>6
months
old).
Children’s
influenza
vaccination
statuses
were
determined
approximately
every
six
months
thereafter.
For
two
years
during
the
study
follow-up,
caregivers
were
contacted
weekly
by
telephone
to
identify
any
acute
respiratory
illness
(ARI)
in
the
enrolled
children,
defined
as
presence
of
>2
of
the
following
symptoms:
fever/feverish,
cough,
sore
throat,
or
runny
nose
with
onset
during
the
preceding
seven
days.
Caregivers
of
children
reporting
ARI
were
encouraged
to
take
the
child
to
the
study
hospital,
on
that
day
or
the
next
day,
for
symptom
verification
and
treatment;
all
had
nasal
and
throat
specimens
collected
during
hospital
visit.
The
specimens
were
tested
by
real-
time
reverse
transcription
PCR
(rRT-PCR)
for
influenza
virus.
All
children
had
2
mL
of
venous
blood
taken
every
six
months
during
the
course
of
the
study
to
measure
influenza
antibody
titers.
Specimens
from
children
who
were
6
months
of
age
at
enrollment
were
tested
as
part
of
this
study.
Laboratory
testing
All
laboratory
testing
was
performed
at
the
Armed
Forces
Research
Institute
of
Medical
Sciences
in
Bangkok,
Thailand.
The
rRT-PCR
assay
was
conducted
on
respiratory
specimens
according
to
the
U.S.
Centers
for
Disease
Control
and
Prevention’s
standard
protocol
(specimens
with
a
cycle
threshold
38
were
classified
as
positive)
(WHO
Collaborating
Centre
for
Influenza,
2009).
The
hemagglutination
inhibition
(HI)
assay
to
measure
influenza
antibody
titers
in
blood
specimens
was
conducted
using
guinea
pig
erythrocytes
according
to
the
World
Health
Organization’s
protocol
(WHO
Collaborating
Center
for
surveillance,
epidemiolo-
gy,
and
control
of
influenza,
2019;
WHO
Global
Influenza
Surveillance
Network,
2011).
Specimens
were
tested
against
a
panel
of
representative
influenza
viruses
circulating
in
Thailand
at
the
time
of
the
specimen
collection:
A/California/07/2009,
A/
Texas/50/2012,
A/Switzerland/9715293/2013,
A/Victoria/361/2011,
B/Brisbane/60/2008,
B/Wisconsin/1/2010,
B/Massachusetts/2/
2012,
and
B/Phuket/3073/2013.
A
set
of
specimens
from
each
child
was
tested
on
the
same
96-well
plate.
All
blood
specimens
were
tested
in
three
batches
to
minimize
inter-batch
variation
as
much
as
possible.
Data
analysis
Children
enrolled
at
6
months
of
age
were
included
in
the
analytical
dataset
if
they
were
born
to
a
mother
who
self-reported
not
having
received
influenza
vaccination
during
pregnancy.
Enrolled
children
provided
at
least
two
blood
specimens
six
months
apart
to
permit
measurement
of
seroconversion.
All
intervals
and
specimens
collected
after
a
child
was
vaccinated
with
influenza
vaccine
were
excluded
from
the
analysis.
Titers
from
the
HI
assay
recorded
as
<10
were
converted
to
5
for
analytical
purposes.
Seroconversion
was
defined
as
a
>4-fold
rise
in
the
HI
titers
for
each
influenza
virus
between
two
consecutive
time
points
with
a
minimum
titer
on
the
second
specimen
of
40.
A
child
could
experience
more
than
one
seroconversion
to
the
same
influenza
virus.
Children’s
age
for
which
the
seroconversion
occurred
was
determined
using
age
at
the
start
of
the
interval
during
which
the
seroconversion
was
measured.
At
each
blood
collection
visit,
we
determined
whether
the
child
had
any
ARI
symptoms,
ascertained
weekly
through
telephone
contact,
during
the
preceding
six
months
up
to
two
weeks
before
blood
collection
since
it
usually
takes
1–2
weeks
for
the
antibodies
to
develop
(Rothbarth
et
al.,
1999;
Kumar
and
Henrickson,
2012).
A
laboratory-confirmed
episode
of
influenza
virus
infection
with
ARI
symptoms
was
defined
as
an
illness
episode
with
symptoms
that
met
the
ARI
definition
and
had
laboratory-confirmed
influenza
virus
infection
(by
rRT-PCR
or
serology).
A
laboratory-confirmed
episode
of
influenza
virus
infection
without
ARI
symptoms
was
defined
as
laboratory-confirmed
influenza
virus
infection
without
any
ARI
symptoms
reported
in
the
preceding
six
months
up
to
two
weeks
before
blood
collection
(by
serology)
or
in
the
proceeding
ten
days
of
respiratory
specimen
collection
(by
rRT-PCR).
The
Chi-Square
test
was
used
to
compare
baseline
demographic
and
household
characteristics
between
healthy
and
high-risk
children.
We
used
a
generalized
linear
model
with
a
log
link
and
a
Poisson
distribution
and
the
natural
log
of
person-time
in
years
as
22
K.
Rungrojcharoenkit
et
al.
/
International
Journal
of
Infectious
Diseases
89
(2019)
21–26

an
offset
to
calculate
incidence
rates
with
95%
confidence
intervals
by
age
and
health
status
(Dilorio,
1991).
Because
influenza
viruses
circulate
perennially
in
Thailand,
there
was
no
need
to
account
for
seasonality
in
the
analysis.
All
data
analyses
were
conducted
using
SAS
software
version
9.0
(SAS
Institute,
Cary,
North
Carolina,
USA).
Results
Characteristics
of
study
participants
Between
August
2011
and
September
2013,
1,149
children
were
enrolled
into
the
cohort;
350
(30%)
children
were
enrolled
at
6
months
of
age.
At
least
two
blood
specimens
were
obtained
from
306
(87%)
eligible
children,
but
40
were
excluded
because
they
received
an
influenza
vaccination
before
the
collection
of
their
second
blood
specimens,
resulting
in
an
analysis
of
266
participants.
Among
these,
147
(55%)
were
healthy
and
119
(45%)
were
considered
high-risk
at
the
time
of
enrollment.
Among
those
included
in
the
analysis,
the
median
age
at
enrollment
was
3
months
(interquartile
range,
2–4).
Healthy
and
high-risk
children
were
similar
with
respect
to
sex,
age
at
enrollment,
daycare
attendance,
distance
from
home
to
hospital,
mother’s
education,
monthly
household
income
of
parents,
and
number
of
siblings
aged
<10
years
old
(Table
1).
However,
healthy
children
were
more
likely
than
high-risk
children
to
have
been
breastfed
at
enrollment
(63%
vs.
50%;
P
value
=
0.04).
None
of
the
mothers
of
children
included
in
this
analysis
reported
receipt
of
influenza
vaccination
during
pregnancies.
Influenza
rRT-PCR
positivity
and
seroconversion
rates
During
the
2-year
follow-up,
266
children
contributed
370
person-years
of
observation
after
excluding
post-vaccination
periods;
healthy
children
contributed
200
person-years
(54%)
and
high-risk
children
contributed
170
person-years
(46%)
(Tabl e
2).
Thirty-two
cases
of
rRT-PCR-confirmed
influenza
virus
infection
were
identified
when
children
came
for
routine
blood
draw
(total
of
706
visits).
Eleven
(35%)
of
the
influenza
viruses
identified
were
influenza
A
(H1N1)pdm09,
12
(38%)
were
influenza
A
(H3N2),
and
9
(28%)
were
influenza
B.
The
overall
rate
of
rRT-PCR-confirmed
influenza
virus
infection
in
this
cohort
was
7
infections
per
100
person-years
(95%
confidence
interval
[CI],
4–11),
with
no
statistical
difference
between
healthy
(8
infections
per
100
person-years,
95%
CI,
5–15)
and
high-risk
children
(6
infections
per
100
person-years,
95%
CI,
3–12;
P
value
=
0.42;
Table
3A).
Among
32
rRT-PCR
positive
cases,
6
(19%)
were
in
children
who
experienced
ARI
symptoms
during
the
ten
days
preceding
the
respiratory
specimen
collection.
There
were
3
(9%)
rRT-PCR
positive
cases
who
had
ARI
symptoms
without
fever,
while
3
(9%)
reported
having
fever
and
cough.
As
measured
through
seroconversions,
there
were
126
episodes
of
influenza
virus
infection
over
370
person-years
of
observation,
resulting
in
a
rate
of
35
infections
per
100
person-years
(95%
CI,
30–42;
Table
3B).
Serology
data
show
a
significantly
higher
rate
of
influenza
virus
infection
than
the
incidence
of
influenza
virus
infection
measured
through
rRT-PCR
(35
vs.
7
infections
per
100
person-years;
P
value
<
0.01).
Among
126
seroconverted
cases,
56
(44%)
experienced
ARI
symptoms
during
the
preceding
six
months
up
to
two
weeks
before
blood
collection
(35
infections
per
100
person-years;
95%
CI,
28–44)
while
the
rest
denied
ARI
symptoms
on
re-interview
(67;
53%)
or
had
only
a
fever
(3
[2%];
33
infections
per
100
person-years;
95%
CI,
25–43;
P
value
=
0.78;
Supplementary
Table
S2).
There
were
6
(5%)
seroconverted
cases
who
had
ARI
symptoms
but
absence
of
fever,
while
44
(35%)
reported
having
fever
and
cough.
The
seroincidence
rate
of
influenza
virus
infection
was
lowest
for
children
between
0
and
6
months
of
age
(23
infections
Table
1
Baseline
characteristics
of
healthy
and
high-risk
children
enrolled
in
the
seroincidence
study,
Bangkok,
Thailand.
Characteristics
All
children
Healthy
High-risk
a
P
value
N
=
266
N
=
147
N
=
119
n
(%)
n
(%)
n
(%)
Male
138
(52)
74
(50)
64
(54)
0.58
Age
at
enrollment
0.32
<2
months
40
(15)
22
(15)
18
(15)
2–3
months
106
(40)
53
(36)
53
(45)
4–6
months
120
(45)
72
(49)
48
(40)
Reported
breast
feeding
at
enrollment
153
(58)
93
(63)
60
(50)
0.04
Attended
daycare
at
enrollment
15
(6)
8
(5)
7
(6)
0.88
Distance
from
home
to
hospital
0.18
<5
km
89
(33)
56
(38)
33
(28)
5–9
km
47
(18)
28
(19)
19
(16)
10–14
km
32
(12)
16
(11)
16
(13)
15
km
98
(37)
47
(32)
51
(43)
Mother
completed
primary
school
205
(77)
120
(82)
85
(71)
0.05
Monthly
household
income
20,000
Thai
Baht
(570
USD)
125
(47)
61
(42)
64
(54)
0.05
Had
1
sibling
aged
<10
years
95
(36)
52
(35)
43
(36)
0.90
a
At
high
risk
for
complications
of
influenza
if
infected
(refer
to
Supplementary
Table
S1
for
detail).
Table
2
Number
of
children
at
the
start
of
each
interval
and
person-time
of
observation.
Age
interval
(month)
b
Number
of
children
at
the
start
of
the
interval
Person-years
All
children
Healthy
High-risk
a
All
Healthy
High-risk
0–6
266
147
119
140
79
64
7–12
178
94
84
94
52
43
13–18
145
76
69
75
39
36
19–24
117
63
54
58
31
27
All
266
147
119
370
200
170
a
At
high
risk
for
complications
of
influenza
if
infected
(refer
to
Supplementary
Table
S1
for
detail).
b
Represents
children
by
age
at
the
start
of
the
interval
during
which
seroconversion
was
measured.
K.
Rungrojcharoenkit
et
al.
/
International
Journal
of
Infectious
Diseases
89
(2019)
21–26
23

per
100
person-years,
95%
CI,
16–33;
Table
3B).
The
overall
rates
did
not
differ
between
healthy
(34
infections
per
100
person-years,
95%
CI,
27–43)
and
high-risk
children
(35
infections
per
100
person-years,
95%
CI,
27–45;
P
value
=
0.88).
Seroincidence
rates
were
higher
for
influenza
A
(H3N2)
(23
infections
per
100
person-
years;
95%
CI,
18–28)
than
for
influenza
A
(H1N1)pdm09
(11
infections
per
100
person-years;
95%
CI,
8-15)
or
influenza
B
(6
infections
per
100
person-years;
95%
CI,
4–10)
viruses
(Table
3C–E).
Discussion
Of
266
children
followed
for
370
person-years,
one
in
three
seroconverted
to
influenza
virus
infection
in
this
study.
We
found
no
difference
in
seroincidence
rates
between
healthy
children
and
children
at
high
risk
of
influenza
complications.
Influenza
virus
infection
without
ARI
symptoms
occurred
in
more
than
half
of
children
in
this
study
and
did
not
differ
between
healthy
and
high-
risk
children.
Table
3
Incidence
of
influenza
virus
infection
per
100
person-years
in
healthy
and
high-risk
children
enrolled
into
the
seroincidence
study,
Bangkok,
Thailand.
A.
rRT-PCR-confirmed
influenza
virus
infection
Age
interval
(month)
b
All
children
Healthy
High-risk
a
Number
Rate
c
(95%
CI)
Number
Rate
c
(95%
CI)
Number
Rate
c
(95%
CI)
0–6
10
4
(2–12)
9
11
(6–21)
1
2
(0–11)
7–12
10
10
(5–19)
7
14
(7–30)
3
7
(2–22)
13–18
9
12
(6–23)
4
11
(4–28)
5
14
(6–33)
19–24
3
5
(1–16)
1
3
(0–23)
2
7
(2–30)
All
32
7
(4–11)
21
8
(5–15)
11
6
(3–12)
P
value
for
comparison
of
healthy
vs
high-risk:
0.42,
P
value
for
age
at
the
start
of
the
interval:
0.22,
P
value
for
interaction
term
between
arm
and
health
status
at
start
of
the
interval:
0.13
B.
Seroincidence
of
any
influenza
virus
Age
interval
(months)
b
All
children
Healthy
High-risk
a
Number
Rate
c
(95%
CI)
Number
Rate
c
(95%
CI)
Number
Rate
c
(95%
CI)
0–6
33
23
(16–33)
19
23
(14–36)
15
23
(14–39)
7–12
39
41
(30–57)
18
37
(23–58)
20
47
(30–72)
13–18
31
41
(29–58)
18
46
(29–74)
13
36
(21–62)
19–24
23
40
(26–60)
13
42
(24–73)
10
37
(20–69)
All
126
35
(30–42)
68
34
(27–43)
58
35
(27–45)
P
value
for
comparison
by
arm:
0.88,
P
value
for
age
at
the
start
of
the
interval:
0.04,
P
value
for
interaction
term
between
arm
and
age
at
start
of
the
interval:
0.78.
C.
Seroincidence
of
influenza
virus
A
(H1N1)
pdm09
Age
interval
(month)
b
All
children
Healthy
High-risk
a
Number
Rate
c
(95%
CI)
Number
Rate
c
(95%
CI)
Number
Rate
c
(95%
CI)
0–6
8
6
(3–11)
4
5
(2–14)
4
6
(2–17)
7–12
13
13
(8–23)
5
10
(4–23)
8
19
(10–37)
13–18
11
14
(8–26)
7
18
(9–38)
4
11
(4–30)
19–24
7
12
(6–26)
4
13
(5–35)
3
11
(4–35)
All
39
11
(8–15)
20
10
(7–16)
19
11
(7–18)
P
value
for
comparison
by
arm:
0.86,
P
value
for
age
at
the
start
of
the
interval:
0.14,
P
value
for
interaction
term
between
arm
and
age
at
start
of
the
interval:
0.57.
D.
Seroincidence
of
influenza
virus
A
(H3N2)
Age
interval
(month)
b
All
children
Healthy
High-risk
a
Number
Rate
c
(95%
CI)
Number
Rate
c
(95%
CI)
Number
Rate
c
(95%
CI)
0–6
22
15
(10–24)
11
14
(8–25)
11
17
(10–31)
7–12
19
20
(13–32)
9
17
(9–33)
10
34
(13–43)
13–18
23
30
(20–46)
14
36
(21–61)
9
25
(13–48)
19–24
16
28
(17–45)
9
36
(21–61)
7
26
(12–55)
All
80
23
(18–28)
43
22
(17–30)
37
22
(16–31)
P
value
for
comparison
by
arm:
0.98,
P
value
for
age
at
the
start
of
the
interval:
0.12,
P
value
for
interaction
term
between
arm
and
age
at
start
of
the
interval:
0.70.
E.
Seroincidence
of
influenza
virus
B
Age
interval
(month)
b
All
children
Healthy
High-risk
a
Number
Rate
c
(95%
CI)
Number
Rate
c
(95%
CI)
Number
Rate
c
(95%
CI)
0–6
6
3
(1–9)
5
6
(3–15)
1
2
(0–11)
7–12
7
7
(3–15)
5
10
(4–23)
2
5
(1–19)
13–18
4
5
(2–14)
2
5
(1–21)
2
6
(1–22)
19–24
8
12
(5–27)
6
19
(9–43)
2
7
(2–30)
All
25
6
(4–10)
18
9
(5–15)
7
4
(2–9)
P
value
for
comparison
by
arm:
0.10,
P
value
for
age
at
the
start
of
the
interval:
0.24,
P
value
for
interaction
term
between
arm
and
age
at
start
of
the
interval:
0.77.
95%
CI,
95%
confidence
interval.
a
At
high
risk
for
complications
of
influenza
if
infected
(refer
to
Supplementary
Table
S1
for
detail).
b
Represents
children
by
age
at
the
start
of
the
interval
during
which
seroconversion
was
measured.
c
Incidence
rates
and
95%
confidence
intervals
by
age
and
health
status
were
calculated
using
a
generalized
linear
model
with
a
log
link
and
a
Poisson
distribution
and
the
natural
log
of
person-time
in
years
as
an
offset.
24
K.
Rungrojcharoenkit
et
al.
/
International
Journal
of
Infectious
Diseases
89
(2019)
21–26

Data
on
seroconversion
in
response
to
influenza
virus
infection
in
Thailand
are
scarce;
no
study
has
reported
seroconversion
data
for
seasonal
influenza
and
most
studies
of
seroconversion
to
influenza
A
(H1N1)pdm09
virus
during
the
2009
influenza
pandemic
focused
on
seroconversion
in
special
populations
(Lerdsamran
et
al.,
2011;
Garg
et
al.,
2014;
Khuntirat
et
al.,
2014;
Simmerman
et
al.,
2011).
For
instance,
using
specimens
collected
in
2009,
Lerdsamran
et
al.
reported
a
substantial
influenza
A
(H1N1)
pdm09
virus-specific
infection
rate
among
healthy
children
aged
<15
years
old
compared
to
healthy
adults
(Lerdsamran
et
al.,
2011).
A
Thai
study
by
Garg
et
al.
reported
influenza
A
(H1N1)pdm09
seroincidence
in
the
population
of
men
who
have
sex
with
men
between
2009–2011
(Garg
et
al.,
2014).
In
our
study,
the
overall
incidence
of
influenza
virus
infection
was
high
(35
infections
per
100
person-years).
Although
it
can
be
difficult
to
compare
results
across
studies
and
settings,
rates
of
seroconversion
to
influenza
virus
infection
in
our
study
were
of
the
same
magnitude
as
rates
reported
from
prior
studies
of
seasonal
influenza
in
older
children,
which
ranged
from
15–42
per
100
person-years
(Horby
et
al.,
2012;
Hayward
et
al.,
2014).
Further,
the
simulated
incidence
among
children
by
stochastic
mechanistic
models
was
estimated
to
range
from
7–29%
annually
based
on
the
USA’s
influenza
serology
data
(Tokars
et
al.,
2018;
Sullivan
et
al.,1993;
Ranjeva
et
al.,
2019).
In
this
study,
we
observed
substantial
proportions
of
individuals
who
seroconverted
but
did
not
report
ARI
symptoms.
An
earlier
study
suggested
that
infected
individuals
with
few
or
no
signs
of
illness
may
shed
infectious
virus
(Foy
et
al.,
1987),
highlighting
the
role
of
infections
without
symptoms
meeting
traditional
respiratory
illness
case
definitions
in
possible
influenza
transmission.
Quanti-
fying
the
proportion
of
serologically
confirmed
infections
without
ARI
symptoms
can
inform
our
understanding
of
influenza
transmission
dynamics.
In
the
PRICE
study,
which
was
a
parental
study
of
this
work,
we
observed
a
higher
incidence
of
influenza
virus
infection
as
determined
by
rRT-PCR
in
healthy
children
compared
to
high-
risk
children
in
the
entire
cohort
(Kittikraisak
et
al.,
2018).
However,
we
did
not
observe
a
similar
difference
in
a
subset
of
very
young
children,
possibly
because
of
the
small
sample
size.
Another
possible
explanation
is
that
younger
children
had
fewer
social
contacts
outside
the
home
(i.e.,
being
nursed
at
home
instead
of
a
daycare/school)
and
thus
fever
opportunities
for
infection.
In
the
current
report,
only
6%
of
children
attended
daycare
at
enrollment,
and
follow-up
ended
when
children
were
around
two
years
of
age.
In
contrast,
13%
of
healthy
children
and
11%
of
high-risk
children
in
the
PRICE
study,
more
than
half
enrolled
after
one
year
of
age,
reported
attending
daycare/
pre-school
at
enrollment.
Our
study
has
several
strengths.
First,
we
collected
sera
every
six
months,
making
it
less
likely
that
our
results
underestimate
seroconversion
because
of
antibody
decline,
which
is
a
concern
when
sera
are
collected
at
longer
intervals
(Hsu
et
al.,
2014).
Second,
we
were
able
to
evaluate
seroconversion
as
a
marker
of
natural
infection
among
a
cohort
of
children
born
to
women
who
had
not
received
influenza
vaccine
during
pregnancy,
thereby
avoiding
maternal
vaccination
effects
that
would
complicate
interpretation
of
sera
results
during
early
infancy.
Third,
high-
risk
children
were
oversampled
in
our
study,
allowing
the
estimation
of
seroincidence
in
this
sub-population.
Several
limitations
should
be
considered
when
interpreting
our
findings.
First,
seroconversions
without
rRT-PCR-confirmed
ARI
may
have
been
associated
with
atypical
presentations
of
influenza
(Polkinghorne
et
al.,
2011;
Silvennoinen
et
al.,
2011;
Blumental
et
al.,
2011;
Launay
et
al.,
2011;
Lochindarat
and
Bunnag,
2011;
Wright
et
al.,1980)
that
may
be
more
common
in
young
infants
and
were
not
captured
by
our
case
definition.
Second,
blood
and
respiratory
specimens
were
not
collected
at
the
same
time
although
we
determined
whether
the
child
had
any
ARI
symptoms
during
the
preceding
six
months
up
to
two
weeks
before
blood
collection.
Third,
although
we
excluded
children
who
were
born
to
vaccinated
mothers,
influenza
virus
infection
among
the
mothers
during
pregnancy
regardless
of
vaccination
status
was
not
assessed
in
our
cohort.
Our
study
shows
that
influenza
virus
infection
is
common
during
the
first
two
years
of
life
among
children
in
Thailand
and
that
a
large
proportion
of
infections
may
not
be
detected
using
the
ARI
case
definition.
Contributions
WK,
SJO,
PS,
TC,
FSD,
KAL
designed
the
research
study.
KR,
WK,
DD,
SJO,
PS,
TC,
CK,
IY,
SF,
LM,
KAL
performed
the
research.
KR
prepared
the
dataset.
WK
and
KAL
analyzed
the
data.
KR
prepared
the
first
draft
of
the
manuscript.
WK,
SJO,
FSD,
KAL
critically
reviewed
and
revised
the
manuscript.
All
authors
reviewed
and
approved
the
manuscript.
Disclaimer
The
findings
and
conclusions
in
this
report
are
those
of
the
authors
and
do
not
necessarily
represent
the
official
position
of
the
U.S.
Centers
for
Disease
Control
and
Prevention,
the
Depart-
ment
of
the
Army,
the
Department
of
Defense,
or
the
U.S.
Government.
Conflict
of
interest
statement
The
authors
have
no
conflicts
of
interest
or
funding
to
disclose.
Acknowledgements
This
project
was
funded
by
U.S.
Centers
for
Disease
Control
and
Prevention
through
cooperative
agreement
5U01GH000152.
We
thank
all
participants
in
this
study.
We
would
like
to
acknowledge
Dr.
Siraporn
Sawasdivorn,
Dr.
Ratanotai
Plubrukarn,
Dr.
Warunee
Punpanich,
Dr.
Varaporn
Sangtawesin,
Dr.
Meera
Khorana,
Dr.
Panida
Srisan,
Dr.
Mukda
Vangveeravong,
Dr.
Naiyana
Neesanan,
Dr.
Orawan
Iam-opas,
Dr.
Sorasak
Lochindarat,
Dr.
Thanarat
Layangool,
Somchit
Ruenthong,
Naruemon
Sassung-
nune,
Jivanan
Chompupuen,
Hatairat
Kunakronadul,
Suthita
Surinchai,
Saranya
Winyawong,
Sudarat
Intranusong,
Patsada
Somkhuntod,
Wimolrat
Thongngern,
Nongnuch
Chanphet,
Rata-
naporn
Nartkul,
Apiradee
Jamtiengtrong,
Bajaree
Chotpitayasu-
nondh,
Suchada
Srisarang,
Jiraporn
Phourai,
Sarayut
Sridontong,
Krongthong
Jirapong,
Chitdej
Jirapong,
Banyen
Kosulvit,
and
Anchalee
Nontongpool
of
the
Queen
Sirikit
National
Institute
of
Child
Health
(Bangkok,
Thailand)
for
their
contribution
to
the
PRICE
study.
We
thank
Dr.
Dean
Erdman
and
Dr.
Eileen
Schneider
of
U.S.
Centers
for
Disease
Control
and
Prevention
for
their
input
in
the
study
protocol.
We
thank
Amonlaya
Chaiyakum,
Duangporn
Limsritrakul,
and
Waraporn
Sakornjun
of
the
Thailand
Ministry
of
Public
Health
-
U.S.
Centers
for
Disease
Control
and
Prevention
Collaboration
(Nonthaburi,
Thailand)
for
their
administrative
and
data
management
assistance
and
Chitchai
Hemachudha,
Parinya
Kruacharoen,
Prachakkra
Panthusiri,
Rewadee
Klinmala,
Suttiman
Wattanasrirote,
Tipawan
Thipwong,
Thipwipha
Phonpakobsin,
Dr.
Butsaya
Thaisomboonsuk,
and
Kittinun
Hussem
of
the
Armed
Forces
Research
Institute
of
Sciences
(Bangkok,
Thailand)
for
their
laboratory
support.
Finally,
we
thank
Dorothy
L.
Southern
for
scientific
communication
advice
on
the
early
draft
of
this
manuscript
and
Dr.
Eduardo
Azziz-Baumgartner,
Dr.
Jerome
Tokars,
Dr.
David
Shay,
and
Dr.
Sandra
Chaves
for
critically
review
the
final
draft
of
this
manuscript.
K.
Rungrojcharoenkit
et
al.
/
International
Journal
of
Infectious
Diseases
89
(2019)
21–26
25

Appendix
A.
Supplementary
data
Supplementary
material
related
to
this
article
can
be
found,
in
the
online
version,
at
doi:https://doi.org/10.1016/j.ijid.2019.08.022.
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