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Dengue Viremia Kinetics in Asymptomatic and Symptomatic infection

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Background: Dengue infection is a global health threat. While symptomatic cases contribute to morbidity and mortality, the majority of infected people are asymptomatic but serve as important reservoir. However, the kinetics of viremia in asymptomatic infections remains unknown. Methods: We enrolled 279 hospital-based symptomatic index cases and quantified dengue virus (DENV) RNA at enrollment and at day of defervescence. To identify asymptomatic cases, 175 household members of index cases were monitored for clinical symptoms during follow-up and blood was taken twice weekly to test for and quantify DENV RNA until cleared. Results: We detected DENV in 13 asymptomatic household members (7.43%). Their DENV serotypes were majoritarily the same as those of their family index cases. The median peak DENV viremia in asymptomatic subjects was lower than that of symptomatic individuals during febrile phase and the viral decay rate was slower in asymptomatic infections. Conclusions: DENV level and kinetics in asymptomatic individuals differed significantly from those of symptomatic cases. Despite the lower viremia, the slower decay rate in asymptomatic infections could lead to their prolonging the infectious reservoir. The improvement of transmission control to prevent such long-lived asymptomatic infections to transmit the DENV is needed.
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Dengue
viremia
kinetics
in
asymptomatic
and
symptomatic
infection
Ponpan
Matangkasombut
a,b,
*,
Kajohnpong
Manopwisedjaroen
a,1
,
Nada
Pitabut
c,d
,
Sasikanya
Thaloengsok
a,2
,
Swangjit
Suraamornkul
e
,
Tawatchai
Yingtaweesak
f
,
Veasna
Duong
g
,
Anavaj
Sakuntabhai
h
,
Richard
Paul
h
,
Pratap
Singhasivanon
i,
**
a
Department
of
Microbiology,
Faculty
of
Science,
Mahidol
University,
Bangkok,
Thailand
b
Systems
Biology
of
Diseases
Research
Unit,
Faculty
of
Science,
Mahidol
University,
Bangkok,
Thailand
c
Ofce
of
Research
Services,
Faculty
of
Tropical
Medicine,
Mahidol
University,
Bangkok,
Thailand
d
Faculty
of
Medicine,
King
Mongkuts
Institute
of
Technology
Ladkrabang,
Bangkok,
Thailand
e
Faculty
of
Medicine,
Vajira
Hospital,
Bangkok,
Thailand
f
Thasongyang
Hospital,
Tak ,
Thailand
g
Virology
Unit,
Institut
Pasteur
du
Cambodge,
Institut
Pasteur
International
Network,
PO
Box
983,
Phnom
Penh,
Cambodia
h
Institut
Pasteur,
Functional
Genetics
of
Infectious
Diseases
Unit,
UMR
2000
(CNRS),
Paris
75015,
France
i
Department
of
Tropical
Hygiene,
Faculty
of
Tropical
Medicine,
Mahidol
University,
Bangkok,
Thailand
A
R
T
I
C
L
E
I
N
F
O
Article
history:
Received
19
August
2020
Received
in
revised
form
22
September
2020
Accepted
22
September
2020
Keywords:
Dengue
Asymptomatic
Inapparent
Virus
kinetics
Viral
decay
Viremia
duration
A
B
S
T
R
A
C
T
Background:
Dengue
infection
is
a
global
health
threat.
While
symptomatic
cases
contribute
to
morbidity
and
mortality,
the
majority
of
infected
people
are
asymptomatic
but
serve
as
an
important
reservoir.
However,
the
kinetics
of
viremia
in
asymptomatic
infections
remains
unknown.
Methods:
We
enrolled
279
hospital-based
symptomatic
index
cases
and
quantied
dengue
virus
(DENV)
RNA
at
enrollment
and
at
the
day
of
defervescence.
To
identify
asymptomatic
cases,
175
household
members
of
index
cases
were
monitored
for
clinical
symptoms
during
follow-up,
and
blood
was
taken
twice
weekly
to
test
for
and
quantify
DENV
RNA
until
cleared.
Results:
We
detected
DENV
in
thirteen
asymptomatic
household
members
(7.43%).
Their
DENV
serotypes
were
primarily
the
same
as
those
of
their
family
index
cases.
The
median
peak
DENV
viremia
in
asymptomatic
subjects
was
lower
than
that
of
symptomatic
individuals
during
the
febrile
phase,
and
the
viral
decay
rate
was
slower
in
asymptomatic
infections.
Conclusions:
DENV
level
and
kinetics
in
asymptomatic
individuals
differed
signicantly
from
those
of
symptomatic
cases.
Despite
the
lower
viremia,
the
slower
decay
rate
in
asymptomatic
infections
could
lead
to
their
prolonging
the
infectious
reservoir.
The
improvement
of
transmission
control
to
prevent
such
long-lived
asymptomatic
infections
from
transmitting
the
DENV
is
needed.
©
2020
The
Authors.
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/).
Introduction
Dengue
is
the
most
important
arthropod-borne
viral
infection
worldwide,
infecting
an
estimated
390
million
people
annually
(Bhatt
et
al.,
2013).
The
incidence
of
dengue
virus
(DENV)
infection
has
been
rising
over
the
last
ve
decades
(Gubler,
2020).
The
outcome
of
DENV
infection
ranges
from
asymptomatic/inapparent
infection,
mild
self-limited
dengue
fever
(DF)
to
severe
dengue
hemorrhagic
fever
(DHF),
with
the
potential
development
of
life-
threatening
dengue
shock
syndrome
(DSS).
However,
the
majority
of
infected
individuals
have
no
or
insufcient
symptoms
to
result
in
a
clinical
presentation;
nevertheless,
they
could
serve
as
a
*
Corresponding
author
at:
Department
of
Microbiology,
Faculty
of
Science,
Mahidol
University,
272
Rama
VI
Road,
Rajatewee,
Bangkok,
10400
Thailand.
**
Corresponding
author:
Department
of
Tropical
Hygiene,
Faculty
of
Tropical
Medicine,
Mahidol
University,
Bangkok,
Thailand.
E-mail
addresses:
ponpan.mat@mahidol.edu
(P.
Matangkasombut),
pratap.sin@mahidol.ac.th
(P.
Singhasivanon).
1
Kajohnpong
Manopwisedjaroen:
Current
afliation
and
address:
Mahidol
vivax
research
unit,
Faculty
of
Tropical
Medicine,
Mahidol
University,
Bangkok,
Thailand
10400.
2
Sasikanya
Thaloengsok:
Current
afliation
and
address:
Department
of
Bacterial
and
Parasitic
Diseases,
Armed
Forces
Research
Institute
of
Medical
Sciences
(AFRIMS),
Bangkok,
Thailand
10400.
https://doi.org/10.1016/j.ijid.2020.09.1446
1201-9712/©
2020
The
Authors.
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
101
(2020)
9097
Contents
lists
available
at
ScienceDirect
International
Journal
of
Infectious
Diseases
journal
homepage:
www.elsevier.com/locate/ijid
signicant
reservoir
for
DENV
transmission
(Endy,
2002;
Endy
et
al.,
2011;
Grange
et
al.,
2014;
ten
Bosch
et
al.,
2018).
The
level
of
DENV
viremia
is
one
of
the
most
important
determinants
of
human
infectiousness
to
mosquitoes
(Nguyen
et
al.,
2013;
Duong
et
al.,
2015),
and
the
duration
of
infection
is
a
crucial
parameter
determining
the
epidemiological
dynamics
of
any
pathogen
and
the
subsequent
R
0
(Anderson
and
May,
1991).
In
symptomatic
DENV
infections,
classical
experimental
infec-
tion
studies
showed
that
DENV
was
infectious
from
two
days
before
and
ve
days
after
illness
onset
(Siler
et
al.,
1926;
Simmons
et
al.,
1931 ;
Nishiura
and
Halstead,
2007).
The
duration
of
infectiousness,
estimated
via
the
success
of
transmission
to
mosquitoes
or
viral
isolation,
was
found
to
range
from
between
17
days
with
a
mean
of
45
days
and
with
longer
durations
in
primary
than
secondary
infections
(Siler
et
al.,
192 6;
Duyen
et
al.,
2020;
Kuberski
et
al.,
1977).
More
recent
studies
using
molecular
detection
of
the
virus
have
revealed
a
similar
duration
of
infection,
with
a
range
lasting
up
to
six
days
post-onset
of
fever
and
with
some
variation
according
to
serotype,
disease
severity,
and
1
vs.
2
infections
(Duyen
et
al.,
2020).
In
experimental
non-human
primate
sylvatic
DENV
infections,
viremia
duration
ranged
from
three
to
ve
days,
depending
on
the
DENV
serotype
(Althouse
et
al.,
2014).
To
date,
the
kinetics
of
DENV
in
individuals
with
asymptomatic
DENV
infections
remains
unknown.
We
conducted
a
cohort
study
to
identify
and
follow
asymp-
tomatic
DENV
infected
individuals,
DF,
and
DHF
patients
prospec-
tively
to
characterize
their
viremia
kinetics.
Asymptomatic
DENV
infected
individuals
were
identied
by
investigating
the
presence
of
DENV
by
Reverse
transcription
PCR
(RT-PCR)
in
household
members
(HHM)
of
symptomatic
DENV
infected
patients
(index
cases)
(Dussart
et
al.,
2012).
Once
identied,
we
followed
these
individuals
prospectively,
monitored
their
symptoms,
and
mea-
sured
the
level
of
DENV
in
blood
samples
twice
a
week
until
DENV
was
cleared
from
the
circulation.
This
knowledge
of
DENV
kinetics
in
asymptomatic
individuals
is
crucial
for
predicting
the
level
and
duration
of
infectiousness
in
this
important
DENV
reservoir.
Figure
1.
Study
design
for
index
cases
and
household
members
investigation.
D1
(day
of
enrollment);
Ddef
(Day
of
defervescence);
Wk2
(two
weeks
after
enrollment);
Mo2
(two
months
after
enrollment);
DENV
PCR
(RT-PCR
of
DENV
result).
P.
Matangkasombut,
K.
Manopwisedjaroen,
N.
Pitabut
et
al.
/
International
Journal
of
Infectious
Diseases
101
(2020)
9097
91
Materials
and
methods
Ethics
statement
The
study
was
approved
by
the
Institutional
Review
Board
of
Faculty
of
Medicine
Vajira
Hospital
(No.015/12)
and
the
Faculty
of
Tropical
Medicine
Mahidol
University
(TMEC
13041).
All
subjects
or
their
legal
guardians
signed
written
informed
consent
before
study
participation.
Study
population
Subjects
were
recruited
from
two
study
sites
in
Bangkok
(Vajira
hospital
and
the
Faculty
of
Tropical
Medicine,
Mahidol
University)
and
one
study
site
in
Tak
province
(Tasongyang
hospital).
Subjects
were
recruited
from
Vajira
hospital
in
20122015,
and
the
Faculty
of
Tropical
Medicine
and
Tak
sites
were
added
in
2015.
A
total
of
279
index
cases
were
enrolled
Index
case
sample
and
data
collection
Demographic
and
clinical
data
of
index
cases
were
collected
with
standardized
case
report
forms.
Blood
samples
were
collected
for
RT-PCR
twice:
on
the
day
of
enrollment
(D1)
and
on
the
day
fever
subsided
(Day
of
defervescence,
Ddef).
Additional
blood
samples
were
collected
once
a
day
during
the
febrile
phase
between
D1
and
Ddef
and
at
2-week
and
2-month
follow-up
time
points
for
a
heme-agglutination
inhibition
(HI)
test
and
other
immunological
studies.
Index
cases
were
classied
into
DF
and
DHF
according
to
WHO
criteria
(World
Health
Organization,
1997)
(Figure
1).
Household
member
samples
and
data
collection
Once
the
index
cases
were
conrmed
for
DENV
infection,
their
HHM
were
enrolled
within
12
days.
Their
body
temperature
and
clinical
symptoms
were
recorded
on
standardized
case
report
forms
throughout
the
follow-up
period.
Blood
samples
were
taken
for
DENV
RT-PCR
on
the
rst
day
of
enrollment
(D1)
and
then
between
2472
hours
later.
The
result
of
DENV
RT-PCR
became
available
within
24
h
after
blood
collection
and
dictated
subsequent
investigation
and
follow-up.
If
DENV
was
detectable
by
RT-PCR,
two
drops
of
blood
were
taken
onto
lter
paper
(Aubry
et
al.,
2012)
every
24
days
until
DENV
become
undetectable
twice
in
a
row
(or
the
subject
was
lost
to
follow-up).
The
clinical
follow-
up
also
ended
when
DENV
was
undetectable
twice
consecutively
by
RT-PCR
(Figure
1).
DENV
detection,
quantication
and
serotype
determination
by
PCR
Serotype-specic
nested
RT-PCR
was
performed
on
all
samples
to
detect
the
presence
of
DENV
RNA
and
determine
the
DENV
serotype.
Quantitative
RT-PCR
(qRT-PCR)
was
used
to
quantify
the
DENV
viral
load
when
possible.
First,
viral
RNA
was
extracted
from
blood
samples
using
the
QIAamp
viral
RNA
mini
kit
(QIAGEN,
Germany)
according
to
the
manufacturer's
instructions
and
stored
at
80
C
until
used.
Serotype-specic
nested
RT-PCR
was
performed
according
to
the
published
protocol
(Lanciotti
et
al.,
1992).
The
serotype
and
quantity
of
DENV
RNA
were
determined
by
the
qRT-PCR
assay,
as
previously
described
(Duong
et
al.,
2015)
(supplementary
Table
1).
Primary/secondary
DENV
infection
determination
In-house
IgM
capture
ELISA
(Duong
et
al.,
2015)
and
HI
assays
were
performed
as
previously
described
(Clarke,
1958).
Paired
plasma
during
acute
infection
and
two
weeks
or
two
months
follow-up
time
points
(when
available)
were
used
for
HI.
Primary
and
secondary
infections
were
determined
using
WHO/TDR
(1997)
criteria
(World
Health
Organization,
1997).
Statistical
analysis
DENV
decay
rate
in
index
cases
was
calculated
based
on
the
DENV
viral
load
on
D1
subtracted
by
that
on
Ddef
and
divided
by
the
number
of
days
between
D1
and
Ddef.
Decay
rates
in
asymptomatic
HHM
were
also
calculated
for
every
time
point
when
RT-PCR
was
performed
on
an
individual.
For
risk
factor
analyses
of
viral
load,
decay
rate,
day
to
defervescence,
and
day
to
viral
clearance,
a
Generalized
Linear
Model
with
Poisson
error
structure
was
tted
to
test
the
association
with
the
following
explanatory
variables:
age
(contin-
uous),
gender
(M/F),
year,
site,
infection
severity
(DHF
vs.
DF)
and
serotype
and
where
indicated,
log10
transformed
viral
load
at
D1.
The
number
of
individuals
included
in
each
analysis
varied:
all
viral
load
samples
were
analyzed
irrespective
of
whether
they
were
subsequently
lost
to
follow-up;
decay
rate
included
only
individu-
als
who
were
not
lost
to
follow-up
and
who
showed
a
decrease
in
viremia
from
D1
to
Ddef
(only
eight
individuals
had
an
increase
in
viremia
from
D1
to
Ddef);
days
to
defervescence
included
all
individuals
not
lost
to
follow-up
irrespective
of
whether
viremia
increased
from
D1
to
Ddef;
day
to
viral
clearance
included
only
those
with
zero
viremia
at
Ddef.
To
assess
whether
there
was
a
difference
in
the
decay
rate
between
asymptomatic
household
members
and
their
index
cases,
a
Generalized
linear
mixed
model
with
Poisson
error
structure
was
tted
to
decay
rate
with
age,
gender,
serotype
(DENV-3
vs.
DENV-
4),
infection
type
(Index
case
vs.
Asymptomatic
HHM)
as
explanatory
variables
and
household
ID
as
the
random
factor.
The
viral
load
at
D1
and
Ddef
of
the
index
cases
and
the
peak
viral
load
of
the
asymptomatic
HHM
were
compared
by
the
Mann-
Whitney
U
test
For
all
analyses,
a
dispersion
parameter
was
estimated
and
used
to
account
for
overdispersion
in
the
data.
Analyses
were
performed
in
Genstat
version
15
(VSN
International,
2017).
Table
1
Index
cases
characteristics.
Number
of
cases
(percentage)
Severity
DF
177
(63.44%)
DHF
102
(36.55%)
Year
2012
31
(11.11%)
2013
118
(42.29%)
2014
16
(5.73%)
2015
114
(40.86%)
Serotype
1
39
(13.98%)
2
38
(13.62%)
3
89
(31.90%)
4
96
(34.40%)
indeterminate
a
17
(6.09%)
Primary/secondary
infection
Primary
22
(8.24%)
Secondary
208
(74.55%)
Indeterminate
49
(17.5%)
Gender
Male
136
(48.75%)
Female
143
(51.25%)
Age
Children(<=15)
88
(31.54%)
Adults
(>15)
191
(68.46%)
a
These
subjects
had
a
positive
result
for
NS1
or
IgM,
but
DENV
RNA
undetectable
by
PCR
and,
therefore,
unable
to
determine
serotype.
92
P.
Matangkasombut,
K.
Manopwisedjaroen,
N.
Pitabut
et
al.
/
International
Journal
of
Infectious
Diseases
101
(2020)
9097
Results
Index
cases
characteristics
290
index
cases
were
initially
enrolled
in
the
study,
of
which
279
cases
had
conrmed
DENV
infection
from
138
households.
DENV
was
detected
by
RT-PCR
in
262
of
these
279
cases
(94.27%).
Of
those
17
with
negative
RT-PCR,
eleven
had
anti-DENV
IgM,
four
had
detectable
NS1,
and
two
had
both
anti-DENV
IgM
and
NS1.
The
majority
of
index
cases
had
DF
(63.44%),
All
four
DENV
serotypes
circulated
during
the
study
years,
with
DENV-4
(34.40%)
and
DENV-3
(31.90%)
being
more
prevalent
than
the
other
serotypes.
An
almost
equal
number
of
male
(48.75%)
and
female
(51.25%)
subjects
were
enrolled,
and
the
majority
were
adults
(68.46%)
(Table
1).
DENV
kinetics
in
index
cases
Overall,
the
mean
viral
load
in
DENV
positive
individuals
on
the
day
of
enrollment
(D1)
was
7.2 4
10
9
viral
copies/mL
(SEM:
3.45
10
9
).
The
time
to
defervescence
ranged
from
1
to
7
days
post
day
of
recruitment
(the
average
time
was
2.28
days,
SEM
0.08).
Of
those
individuals
that
cleared
their
viral
load
completely
by
the
day
of
defervescence,
the
mean
time
to
clearance
was
2.77
days
(SEM
0.15).
DENV
viral
load
on
D1
was
higher
in
DHF
than
DF
(Relative
Risk
(RR)
=
5.88,
P
<
0.001),
decreased
with
age
(RR
=
0.92,
P
=
0.003),
was
lower
in
male
than
female
gender
(RR
=
0.31,
P
<
0.001)
and
DENV-1
(RR
=
0.25,
P
=
0.001),
DENV-2
(RR
=
0.08,
P
=
0.002),
and
DENV-4
(RR
=
0.06,
P
=
0.006)
as
compared
to
DENV-3
(Table
2,
top).
The
full
minimum
adequate
model
explained
29.2%
of
the
variation
in
viral
load.
DENV
decay
rate
(from
D1
to
Ddef)
was
faster
in
DHF
than
DF
(RR
=
1.4 4 ,
P
=
0.004)
and
DENV-1
(RR
=
1.43,
P
=
0.014)
as
compared
to
DENV-3
(Table
2,
middle)
and
with
an
increased
viral
load
on
D1
(RR
=
6.34,
P
<
0.001).
The
full
minimum
adequate
model
explained
94.7%
of
the
variation
in
the
decay
rate.
Time
taken
to
defervescence
decreased
with
age
(RR
=
0.99,
P
=
0.003)
and
increased
with
viral
load
at
D1
(RR
=
1.12 ,
P
<
0.001)
and
for
DENV-4
(RR
=
1.53,
P
<
0.001)
as
compared
to
DENV-3
(Table
2,
bottom).
The
full
minimum
adequate
model
explained
27.4%
of
the
variation
in
days
to
defervescence.
A
similar
result
was
found
when
using
only
those
individuals
who
had
a
completely
cleared
viremia
on
the
day
of
defervescence.
Rate
of
asymptomatic
DENV
infections
in
household
members
of
dengue
index
cases
Overall,
thirteen
subjects
of
175
HHM
(7.43%)
from
the
138
households
investigated
had
asymptomatic
DENV
infections
(hereon
called
"asymptomatic
HHM")
as
determined
by
the
presence
of
DENV
RNA
by
RT-PCR
and
absence
of
symptoms
during
the
follow-up
period.
These
thirteen
asymptomatic
HHM
were
from
eleven
households
out
of
the
138
households
investigated
(7.97%).
There
was
an
additional
one
HHM
with
DENV
viremia
that
subsequently
developed
symptoms
(pre-
symptomatic)
in
Tak.
A
further
two
HHM
had
had
a
recent
clinical
dengue
infection
within
two
weeks
before
their
family
index
cases
were
diagnosed.
Overall,
the
attack
rate
with
more
than
one
infection
in
a
household
(including
asymptomatic,
pre-symptom-
atic,
and
recent
DENV
infection)
was
14/138
households
(10.14%).
The
proportion
of
households
with
asymptomatic
HHM
was
6/53
(11.32%)
in
Bangkok
and
5/85
(5.88%)
in
Tak
(Figure
2).
Table
2
Factors
associated
with
index
case
DENV
kinetics.
A.
Day
1
Viral
load
N
Mean
Log
10
Viremia
(SEM)
RR
(95%
CI)
P-value
Age
(years)
222
7.31
(0.14)
0.92
(0.870.97)
0.003
Gender
F
113
7.12
(0.21)
Ref
M
109
7.51
(0.18)
0.31
(0.160.62)
<0.001
Severity
DF
142
7.34
(0.17)
Ref
DHF
80
7.2 7
(0.23)
5.88
(2.9211.82)
<0.001
Serotype
1
38
7.11
(0.40)
0.25
(0.110.57)
0.001
2
31
6.82
(0.40)
0.08
(0.020.40)
0.002
3
80
7.54
(0.24)
Ref
4
73
7.39
(0.17)
0.06
(0.010.43)
0.006
B.
Decay
Rate
N
Mean
(SEM)
decay
rate/day
RR
P-value
Severity
DF
114
7.47
(6.83)
Ref
DHF
69
7.63
(7.03)
1.4 4
(1.121.84)
0.004
Serotype
1
30
7.7 1
(7.13)
1.43
(1.081.90)
0.014
2
25
7.66
(7.13)
0.32
(0.792.08)
0.319
3
69
7.55
(6.97)
Ref
4
59
7.30
(6.92)
0.18
(0.241.32)
0.182
Log10
Viral
load
D
1
183
7.54
(6.90)
6.34
(5.497.32)
<0.001
C.
Days
to
Defervescence
N
Mean
(SEM)
days
RR
P-value
Age
(years)
225
2.38
(0.08)
0.990
(0.9860.997)
0.003
Serotype
1
32
2.24
(0.18)
1.13
(0.931.37 )
0.21
2
35
2.07
(0.19)
1.0 4
(0.841.29)
0.69
3
79
1.98
(0.11)
Ref
4
79
3.03
(0.16)
1.53
(1.311.78)
<.001
Log10
Viral
load
D
1
225
2.38
(0.08)
1.12
(1.081.16)
<.001
Shown
in
this
table
are
the
number
of
samples
analyzed
for
each
of
the
signicant
risk
factors,
the
Relative
Risk
(RR)
and
associated
P-value
and
the
dependent
variable
estimate
with
standard
error
from
the
nal
t
in
the
multivariate
log-linear
regression
(see
Methods).
P.
Matangkasombut,
K.
Manopwisedjaroen,
N.
Pitabut
et
al.
/
International
Journal
of
Infectious
Diseases
101
(2020)
9097
93
Characteristics
of
subjects
with
asymptomatic
dengue
infection
and
their
family
index
cases
The
characteristics
of
each
of
the
thirteen
asymptomatic
HHM
and
their
family
index
cases
are
shown
in
Supplementary
Table
2.
There
were
two
households
from
the
Bangkok
site
that
had
two
asymptomatic
HHM
in
the
same
house.
Most
but
not
all
of
the
asymptomatic
HHM
9/13
(69.23%)
harbored
the
same
DENV
serotypes
as
their
family
index
cases.
Interestingly,
two
asymp-
tomatic
HHM
(H1
and
H2)
had
co-infection
with
two
DENV
serotypes
(DENV-3
and
-4),
while
their
family
index
case
was
infected
with
only
DENV-4.
An
additional
asymptomatic
HHM
(H9)
had
a
different
DENV
serotype
from
the
family
index
case;
there
was
one
index
case
(family
of
H10)
for
whom
we
do
not
know
the
DENV
serotype.
Most
family
index
cases
were
male
9/11
(81.82%),
while
5/13
(38.46%)
of
asymptomatic
HHM
were
male.
Most,
10/11
(90.90%),
family
index
cases
had
DF,
and
only
one
had
DHF.
The
kinetics
of
DENV
viremia
in
asymptomatic
infections
Overall,
the
DENV
kinetics
varied
widely
among
individuals
(Supplementary
Fig.
1).
While
some
asymptomatic
HHM
had
high
viremia
but
rapidly
cleared
(H1,
H2,
H6,
H11),
others
had
lower
viremia
that
lasted
longer
(H3,
H4).
H9
likely
had
a
very
low
viral
load
below
the
detection
limit
of
qRT-PCR,
but
detectable
by
nested
PCR.
Unfortunately,
several
subjects
were
lost
to
follow-up
before
the
virus
was
cleared
from
the
circulation.
Taken
together,
the
DENV
kinetics
in
asymptomatic
HHM
are
variable,
but
DENV
viremia
could
persist
up
to
12
weeks
after
the
detection
of
index
cases
in
the
household.
Factors
affecting
DENV
kinetics
in
asymptomatic
infections
Excluding
the
single
instance
of
a
DENV-1
infection,
we
assessed
risk
factors
associated
with
viral
load,
decay,
and
clearance
rate.
The
mean
maximum
viral
load
was
3.89
10
6
viral
copies/mL
(SEM
1.3 5
10
6
);
there
was
no
association
with
any
explanatory
variables
(age,
gender,
serotype,
mixed
or
single
serotype
infection:
P
>
0.05).
The
time
needed
for
DENV
clearance
from
the
maximum
(measured)
viral
load
was
found
to
decrease
with
increasing
maximum
viral
load
(χ
21
=
5.54,
P
=
0.019)
and
to
be
also
faster
in
mixed
serotype
infections
(Single
serotype
infections:
Mean
6.4
days
SEM
1.5;
Mixed
serotype
infections:
3.0
days
SEM
0.7.
χ
21
=
5.02,
P
=
0.025).
Comparing
asymptomatic
HHM
and
index
case
viral
kinetics
The
peak
viremias
of
asymptomatic
DENV
infections
were
lower
than
those
of
index
cases
at
the
D1
when
patients
were
still
febrile.
At
Ddef,
index
cases
viral
loads
dropped
markedly
from
D1
Figure
2.
Numbers
of
index
cases
and
household
members
investigated
and
numbers
of
asymptomatic
dengue
viremia
in
Bangkok
and
Tak
study
sites.
Figure
3.
Dengue
viral
load
in
asymptomatic
dengue
infected
HHM
compared
to
index
cases.
The
peak
viral
load
of
asymptomatic
dengue
infected
HHM
(asymptomatic
(peak))
was
compared
to
the
viral
load
of
index
cases
at
the
day
of
enrollment
(D1)
and
day
defervescence
(Ddef).
Mann-Whitney
was
used
to
compare
two
groups
indicated,
*p
<
0.05,
**p
<
0.01,
***p
<
0.001.
94
P.
Matangkasombut,
K.
Manopwisedjaroen,
N.
Pitabut
et
al.
/
International
Journal
of
Infectious
Diseases
101
(2020)
9097
and
were
even
lower
than
those
of
asymptomatic
HHM
peak
viral
load
(Figure
3).
Asymptomatic
infections
were
associated
with
a
slower
decay
rate
than
index
cases
(Index
case:
mean
4.31
10
8
/
day
SEM:
2.91
10
8
;
Asymptomatic
HHM:
5.32
10
5
/
day,
SEM
2.89
x
10
5
.
χ21
=72.0,
P=0.003)
(Figure
4).
Discussion
A
DENFREE
cohort
of
DENV
infected
patients
with
their
household
members
in
Thailand
was
established
to
identify
asymptomatic
DENV
infections
to
provide
the
rst
description
of
viral
kinetics
within
asymptomatic
infections
and
compare
them
with
that
in
symptomatic
clinical
presentations
from
the
vicinity
and/or
the
same
household.
In
the
symptomatic
index
cases
with
a
measurable
virus
at
enrollment
(D1),
viremias
were
generally
higher
than
those
previously
reported
(Duyen
et
al.,
2020;
Yeh
et
al.,
2020),
but
time
to
defervescence
and/or
viral
clearance
signicantly
faster
(Duyen
et
al.,
2020;
Yeh
et
al.,
2020;
Gubler
et
al.,
1978;
Gubler
et
al.,
1979;
Vaughn
et
al.,
1997;
Libraty
et
al.,
2002).
Signicant
variation
within
viral
load
and
time
to
both
defervescence
and
viral
clearance
was
observed.
Notably,
patients
with
DHF
and
DENV-3
had
a
higher
viral
load,
and
those
who
were
male
and
older
had
lower
viral
loads.
A
trend
for
increased
viremia
with
disease
severity
has
been
observed
previously
(Perdomo-
Celis
et
al.,
2017),
and
variation
amongst
serotypes
has
also
been
noted
(Duyen
et
al.,
2020).
Previous
studies
have
repeatedly
found
shorter
durations
of
infection
in
secondary
infections
as
compared
to
primary
infections
but
which
also
had
higher
viremias
(Kuberski
et
al.,
1977;
Duyen
et
al.,
2020;
Yeh
et
al.,
2020;
Libraty
et
al.,
2002).
In
our
study,
we
had
too
few
primary
infections
to
make
a
reasonable
comparison.
The
viral
decay
rate
from
enrollment
to
defervescence
was
faster
in
patients
with
DHF
than
those
with
a
higher
viral
load
at
enrollment.
Despite
this,
time
to
defervescence
was
still
longer
with
higher
enrollment
viral
load.
From
index
case
houses,
7.43%
of
HHM
of
index
cases
had
DENV
viremia
without
any
symptoms.
Most
of
these
asymptomatic
HHM
had
the
same
DENV
serotypes
as
their
family
index
cases.
The
kinetics
of
DENV
of
these
asymptomatic
HHM
were
highly
variable
among
individuals.
There
were
no
factors
associated
with
the
maximum
measured
viral
load.
A
higher
decay
rate
was
found
in
DENV-4
infections,
and
time
to
viral
clearance
was
faster
with
increasing
viral
load
and
mixed
serotype
infections.
Thus,
in
both
index
cases
and
asymptomatic
infections,
high
viral
load
was
associated
with
more
rapid
viral
decay,
although
this
did
not
lead
to
clearance
at
defervescence
in
symptomatic
cases.
Signicant
among
serotype
differences
in
viral
kinetics
were
observed
here
as
often
before
(Duyen
et
al.,
2020),
and
both
among
and
within-
serotype
differences
in
the
immune
response
have
been
recently
highlighted
(Katzelnick
et
al.,
2015),
albeit
not
in
the
context
of
asymptomatic
infections.
Although
the
exact
duration
of
viremia
is
difcult
to
estimate,
the
viremia
lasted
up
to
two
weeks
in
some
of
the
asymptomatic
infections,
and
the
decay
rate
was
slower
than
that
of
index
cases.
Similarly,
compared
with
their
index
cases,
the
maximum
level
of
viremia
in
asymptomatic
HHM
was
lower
than
that
of
index
cases
at
enrollment
but
higher
than
that
on
the
day
of
defervescence,
consistent
with
the
observed
slower
overall
decay
rate
in
asymptomatic
infections.
This
observation
supports
previous
studies
that
found
lower
viremia
in
asymptomatic
infections
than
those
of
symptomatic
infections
(Duong
et
al.,
2015;
Dussart
et
al.,
2012;
Sowath
et
al.,
2019
Overall,
the
kinetics
of
asymptomatic
infections
differ
from
symptomatic
infections
in
the
magnitude
of
the
viremia
and
the
rate
of
clearance,
suggesting
these
infections
last
longer
but
with
a
lower
viremia.
We
and
others
have
previously
described
fundamental
immunological
differences
in
the
immune
responses
associated
with
symptomatic
and
asymptomatic
infections
(García
et
al.,
2010;
Simon-Lorière
et
al.,
2017;
Halstead
and
ORourke,
1977).
A
polymorphism
in
the
FcgRIIA
was
found
to
be
associated
with
inapparent
infection
vs.
DF
or
DHF
in
the
Cuban
population
(García
et
al.,
2010).
Asymptomatic
DENV
infected
individuals
have
been
found
to
have
increased
T
cell
responses
with
feedback
regulation
when
compared
to
symptomatic
counterparts
(Simon-Lorière
et
al.,
2017).
Classically
secondary
infections
are
associated
with
more
severe
disease
due
to
the
phenomenon
of
ADE
and/or
cross-reactive
T
cells
(Halstead
and
ORourke,
1977),
but
whether
or
not
this
leads
to
a
decreased
risk
of
an
infection
being
inapparent
remains
moot
(Grange
et
al.,
2014;
Clapham
and
Cummings,
2020).
Post-secondary
infections
will
likely
induce
different
immune
responsiveness
and
have
also
been
found
to
impact
upon
inapparent
rates
(Olkowski
et
al.,
2013),
which
could
introduce
additional
variability
in
the
viral
kinetics
in
both
asymptomatic
and
symptomatic
infections.
In
our
study,
however,
the
vast
majority
of
infections
were
secondary,
although
it
is
notable
that
increasing
age
had
a
signicant
impact
on
viral
clearance
rates,
suggesting
a
potential
impact
of
post-
secondary
infections.
More
recently,
the
importance
of
the
interplay
between
viral
genotype
within
serotype
and
the
interaction
with
the
immune
response
has
been
found
to
be
signicant
(Katzelnick
et
al.,
2015;
OhAinle
et
al.,
2020),
thereby
introducing
additional
variability
that
we
can
not
capture
in
our
study.
We
have
previously
shown
that
asymptomatic
infections
are
as,
if
not
more,
infectious
to
mosquitoes
than
symptomatic
infections
(Duong
et
al.,
2015).
The
likelihood
that
such
asymptomatic
infections
also
last
longer
does
suggest
that
their
epidemiological
contribution
is
even
more
important.
In
symptomatic
dengue
infections,
the
level
of
dengue
viremia
was
shown
to
be
the
most
critical
factor
for
transmission
to
mosquitoes
(Duong
et
al.,
2015).
Thus,
both
those
asymptomatic
cases
with
higher
but
shorter-lived
viremia
and
those
with
lower
but
longer-lasting
viremia
could
contribute
to
transmitting
the
disease.
A
wide
range
of
different
epidemiological
studies
has
attempted
to
ascertain
the
extent
of
asymptomatic
infections
and
associated
risk
factors
(Endy,
2002;
Endy
et
al.,
2011;
Grange
et
al.,
2014;
Gordon
et
al.,
2013;
Balmaseda
et
al.,
2010;
Montoya
Figure
4.
Dengue
viral
load
decay
rate
by
day
for
asymptomatic
HHM
infections
and
index
cases
from
the
day
of
recruitment
(D1)
to
the
day
of
defervescence
(Ddef).
The
decay
rate
is
log
(10)
transformed
for
visual
clarity.
P.
Matangkasombut,
K.
Manopwisedjaroen,
N.
Pitabut
et
al.
/
International
Journal
of
Infectious
Diseases
101
(2020)
9097
95
et
al.,
2013;
Morrison
et
al.,
2010).
It
is
widely
agreed
that
inapparent,
sub-clinical
infections
are
prevalent,
and
thus
this
silent
infectious
reservoir
will
be
a
signicant
contributor
to
transmission.
That
we
observed
asymptomatic
infections
lasting
for
two
weeks
will
prove
particularly
problematic
in
preventing
their
role
as
a
reservoir
through
traditional
fumigating
approaches
around
index
cases:
mosquito
re-invasion
of
a
fumigated
neighborhood
occurs
far
quicker
than
such
slow-
decaying
infections.
A
more
pro-active
program
encouraging
individual
level
protection
of
household
members
of
index
cases
from
being
bitten
by
mosquitoes
is
likely
the
only
reasonable
approach
to
reduce
any
epidemiological
contribution
of
poten-
tially
infected
asymptomatic
HHM.
The
relatively
low
percentage
of
infected
HHM
found
in
this
study
is
likely
underestimated,
further
underscoring
the
futility
of
single
time
point
surveys
to
see
whether
HHM
of
index
cases
are
infected:
the
variable
intrinsic
incubation
period
undermines
the
utility
of
active
"case"
detection.
In
conclusion,
his
work
not
only
the
signicant
role
that
asymptomatic
infections
can
play
in
DENV
epidemiology
but
also
emphasizes
the
need
for
alternative
strategies
to
prevent
infected
individuals
from
spreading
the
virus
in
the
course
of
their
daily
mobility.
Funding
This
study
was
supported
by
the
European
Union
Seventh
Framework
Program
(FP7/20072013)
[under
Grant
Agreement
#282,378
(DENFREE)]
(to
AS,
RP,
PS,
PM)
and
a
National
Research
University
grant
(Mahidol,
to
AS,
PM).
The
funders
had
no
role
in
study
design,
data
collection,
analysis,
decision
to
publish,
or
manuscript
preparation.
Authors
contribution
RP,
AS,
PS,
PM
designed
the
study;
KM,
ST
performed
experi-
ments;
NP
clinical
data
collection
and
cohort
management;
PM,
VD
supervised
experiments;
PM,
RP
analyzed
data;
SS,
TY
oversaw
patient
recruitment
and
clinical
data
collection;
PM
oversaw
cohort
sample
collection;
RP,
AS
coordinated
the
multinational
DENFREE
project;
PS,
PM
managed
DENFREE:
Thailand
cohort;
PM,
RP
wrote
the
manuscript;
PM,
VD,
RP
edited
the
manuscript,
and
all
authors
read
and
approved
the
nal
manuscript.
Conict
of
interest
statement
All
authors
declared
no
conict
of
interest.
Acknowledgments
We
thank
Wilawan
Chan-in
for
assistance
with
gure
preparation.
We
thank
all
subjects
and
their
families
for
their
participation
in
this
study.
We
are
grateful
to
the
nursing
team
for
assistance
in
subject
recruitment
and
follow-up.
We
thank
the
Faculty
of
Science,
Mahidol
University,
Central
Instrument
Facility,
Thailand
Research
Fund
(TRG5880121),
and
Anandamahidol
foundation
for
PM
support.
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.2020.09.1446.
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