Epidemiological and etiological characteristics of hand, foot, and mouth disease in Ningbo, China, 2008-2011

Department of Viral Research, Ningbo Center of Disease Control and Prevention, Ningbo 315010, China.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology (Impact Factor: 3.02). 05/2012; 54(4):342-8. DOI: 10.1016/j.jcv.2012.04.021
Source: PubMed
ABSTRACT
Outbreaks of hand, foot, and mouth disease (HFMD) in central China have caused public health concerns since 2007. It is of particular public health significance to update epidemiology of HFMD in port cities.
To investigate epidemical, etiological and clinical characteristics of HFMD in Ningbo, China, from 2008 to 2011.
From May 2008 to December 2011, a total of 37,404 HFMD cases including 196 severe and 12 fatal cases were investigated. Human enteroviruses from 2360 cases were determined by real-time RT-PCR. The VP1 gene of EV71 from 78 cases and CA16 from 21 cases, the VP4 gene from 28 cases, and full-length genomes of 10 isolates were analyzed. Neutralizing antibodies were evaluated in 258 healthy subjects. Parameters associated with severe HFMD were evaluated.
Annual incidence of HFMD was 3066.8/100,000 in the population of ≤5 years. EV71 C4a, CA16 B1, and other enteroviruses accounted for 63.7%, 24.0% and 12.3%, respectively. The genomes of EV71 from fatal and non-fatal cases were nearly identical. The positive rates of neutralizing antibody to EV71 increased from 13.5% to 67.6% in 1- to 5-year healthy groups. The neutralizing antibody to CA16 B1 isolate was negative. EV71, exposure history and certain early manifestations including fever, vomiting, limb exanthema and peripheral neutrophil ratio were significantly associated with HFMD severity.
HFMD mainly caused by EV71 C4a and CA16 B1 is seriously epidemic in Ningbo. Future emphasis should be paid on EV71 immuno-prophylaxis and early identification of severe cases upon the etiological and clinical characteristics.

Full-text

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Journal
of
Clinical
Virology
54 (2012) 342–
348
Contents
lists
available
at
SciVerse
ScienceDirect
Journal
of
Clinical
Virology
jo
u
r
n
al
hom
epage:
www.elsevier.com/locate/jcv
Epidemiological
and
etiological
characteristics
of
hand,
foot,
and
mouth
disease
in
Ningbo,
China,
2008–2011
Hongxia
Ni
a,b
,
Bo
Yi
a
,
Jianhua
Yin
b
,
Ting
Fang
a
,
Tianfeng
He
a
,
Yan
Du
b
,
Jin
Wang
a
,
Hongwei
Zhang
b
,
Lei
Xie
a
,
Yibo
Ding
b
,
Wenzhen
Gu
a
,
Shu
Zhang
a
,
Yifang
Han
b
,
Hongjun
Dong
a
,
Tong
Su
b
,
Guozhang
Xu
a,
,
Guangwen
Cao
b,∗∗
a
Department
of
Viral
Research,
Ningbo
Center
of
Disease
Control
and
Prevention,
Ningbo
315010,
China
b
Department
of
Epidemiology,
Shanghai
Key
Laboratory
of
Medical
Biodefense,
Second
Military
Medical
University,
Shanghai
200433,
China
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
11
January
2012
Received
in
revised
form
25
April
2012
Accepted
30
April
2012
Keywords:
Hand,
foot,
and
mouth
disease
Human
enterovirus
Molecular
epidemiology
Neutralizing
antibody
a
b
s
t
r
a
c
t
Background:
Outbreaks
of
hand,
foot,
and
mouth
disease
(HFMD)
in
central
China
have
caused
public
health
concerns
since
2007.
It
is
of
particular
public
health
significance
to
update
epidemiology
of
HFMD
in
port
cities.
Objective:
To
investigate
epidemical,
etiological
and
clinical
characteristics
of
HFMD
in
Ningbo,
China,
from
2008
to
2011.
Study
design:
From
May
2008
to
December
2011,
a
total
of
37,404
HFMD
cases
including
196
severe
and
12
fatal
cases
were
investigated.
Human
enteroviruses
from
2360
cases
were
determined
by
real-time
RT-PCR.
The
VP1
gene
of
EV71
from
78
cases
and
CA16
from
21
cases,
the
VP4
gene
from
28
cases,
and
full-length
genomes
of
10
isolates
were
analyzed.
Neutralizing
antibodies
were
evaluated
in
258
healthy
subjects.
Parameters
associated
with
severe
HFMD
were
evaluated.
Results:
Annual
incidence
of
HFMD
was
3066.8/100,000
in
the
population
of
5
years.
EV71
C4a,
CA16
B1,
and
other
enteroviruses
accounted
for
63.7%,
24.0%
and
12.3%,
respectively.
The
genomes
of
EV71
from
fatal
and
non-fatal
cases
were
nearly
identical.
The
positive
rates
of
neutralizing
antibody
to
EV71
increased
from
13.5%
to
67.6%
in
1-
to
5-year
healthy
groups.
The
neutralizing
antibody
to
CA16
B1
isolate
was
negative.
EV71,
exposure
history
and
certain
early
manifestations
including
fever,
vomiting,
limb
exanthema
and
peripheral
neutrophil
ratio
were
significantly
associated
with
HFMD
severity.
Conclusions:
HFMD
mainly
caused
by
EV71
C4a
and
CA16
B1
is
seriously
epidemic
in
Ningbo.
Future
emphasis
should
be
paid
on
EV71
immuno-prophylaxis
and
early
identification
of
severe
cases
upon
the
etiological
and
clinical
characteristics.
© 2012 Elsevier B.V. All rights reserved.
1.
Background
Hand,
foot,
and
mouth
disease
(HFMD),
a
communicable
disease
usually
affecting
young
children,
is
generally
a
febrile
exanthematous
disease
but
can
cause
life-threatening
clinical
man-
ifestations
such
as
brainstem
encephalitis
and
cardiopulmonary
complications.
1
Human
enterovirus
71
(EV71)
and
coxsackievirus
A16
(CA16)
are
the
most
common
causes
of
HFMD,
although
sev-
eral
other
human
EVs
including
coxsackieviruses
CA2,
CA4–CA7,
CA10,
CA12,
and
CB1–CB5
can
also
cause
the
disease.
2–6
EV71
is
a
major
etiological
agent
and
frequently
associated
with
severe,
sometimes
fatal
neurological
manifestations.
According
to
Corresponding
author.
Tel.:
+86
574
87680157.
∗∗
Corresponding
author.
Tel.:
+86
21
81871060.
E-mail
addresses:
xugz@nbcdc.org.cn
(G.
Xu),
gcao@smmu.edu.cn,
caoguangwen@yahoo.com
(G.
Cao).
phylogenetic
analysis
of
the
structural
VP1
gene,
EV71
is
classified
into
A,
B
and
C
lineages.
Group
A
has
one
member
(prototype)
while
the
latter
two
are
further
divided
into
subgroups,
B1–B5
and
C1–C5.
Different
groups
and
subgroups
of
EV71
have
distinct
geographic
distributions.
7
Recent
outbreaks
of
HFMD
in
East
and
Southeast
Asia
have
caused
major
public
health
concerns
worldwide.
8–14
Several
outbreaks
in
central
China
have
been
reported
since
2007.
10–14
HFMD
has
been
classified
as
a
notifiable
disease
in
China
since
an
EV71-caused
HFMD
outbreak
occurred
in
Fuyang,
Anhui
province
in
early
2008.
10
EV71
circulating
in
mainland
China
dur-
ing
this
epidemic
wave
is
the
most
recent
descendant
of
subgroup
C4
(C4a).
15
The
occurrence
of
HFMD
has
seasonal
characteristics
and
is
often
associated
with
climate
changes.
16
However,
little
is
known
about
the
epidemiology
of
HFMD
in
the
eastern
coastal
region
of
China
from
2008
to
2011.
Ningbo
is
located
close
to
eastern
coast
of
China
and
south-east
corner
of
the
Yangtze
River,
and
belongs
to
a
north
subtropical
monsoon
climate
zone
with
an
annual
average
1386-6532/$
see
front
matter ©
2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jcv.2012.04.021
Page 1
H.
Ni
et
al.
/
Journal
of
Clinical
Virology
54 (2012) 342–
348 343
temperature
of
16.4
C.
It
is
of
particular
public
health
significance
to
update
molecular
epidemiology
of
HFMD
in
Ningbo,
one
of
the
largest
port
cities
in
China,
with
a
population
of
approximately
7,600,000.
2.
Objectives
The
purpose
of
this
study
was
to
investigate
epidemiological,
etiological
and
clinical
characteristics
of
HFMD
in
Ningbo,
China,
from
2008
to
2011.
3.
Study
design
3.1.
Clinical
information
and
specimen
collection
All
HFMD
cases
were
reported
to
the
Ningbo
Center
for
Disease
Control
and
Prevention
(CDC)
via
the
National
Notifiable
Disease
Reporting
System
(NNDRS)
by
medical
workers
since
May
2008.
All
patients
were
diagnosed
according
to
the
criteria
of
Chinese
Ministry
of
Health
(http://www.moh.gov.cn/publicfiles/business/
htmlfiles/mohyzs/s3585/200812/38494.htm).
Briefly,
mild
case
was
characterized
by
mucocutaneous
papulovesicular
lesions
on
the
hands,
feet,
mouth,
and
buttocks
with
or
without
fever,
which
usually
resolved
spontaneously
within
a
week;
severe
case
was
characterized
by
a
febrile
exanthematous
disease
with
encephali-
tis
and/or
cardiopulmonary
complications.
Laboratory-confirmed
case
met
one
of
the
three
criteria:
(1)
positive
EV71
and/or
CA16
specific-nucleic
acids;
(2)
isolation
of
EV71,
CA16
or
other
EVs;
and
(3)
4-fold
increase
in
the
titers
of
neutralized
antibodies
to
EV71,
CA16
or
other
EVs
in
the
recovery
phase
than
in
the
acute
phase.
The
patients’
guardians
were
interviewed
by
the
CDC
staff
using
a
structured
questionnaire
to
request
data
including
sociodemo-
graphic
and
clinical
information.
The
data
were
verified
by
checking
against
medical
records.
Throat
or
rectal
swabs
and/or
stool
spec-
imens
of
randomly
selected
mild
cases
from
sentinel
hospitals
of
each
district
at
different
seasons
and
all
available
severe
and
fatal
cases
were
collected
by
medical
workers
and
sent
to
our
laborato-
ries
on
ice
for
etiological
assays.
3.2.
Molecular
typing
of
EVs
RNA
was
extracted
from
clinical
specimen
using
the
QIAamp
Viral
RNA
Mini
Kit
according
to
the
manufacturer’s
instruction
(Qiagen,
Hilden,
Germany).
All
RNA
samples
were
examined
by
real-time
RT-PCR
with
the
primers
and
probes
of
Pan-EVs,
EV71,
and
CA16
using
One-Step
PrimerScript
RT-PCR
kits
(TaKaRa,
Dalian,
China).
Using
ABI
Primer
Express
3.0
software,
we
designed
the
Pan-
EV
primer–probe
set
in
the
5
conserved
region
of
EV
genomes
and
the
VP1-specific
primer–probe
sets
for
EV71
and
CA16
from
the
genome
sequences
retrieved
from
GenBank.
The
specimens
only
positive
for
Pan-EVs
were
randomly
selected,
subjected
for
one-
step
RT-PCR
to
amplify
the
VP4
genes,
and
identified
by
sequence
analysis
as
previously
described.
17
Full-length
viral
genomes
were
examined
by
sequencing
viruses
isolated
using
cell
culture.
Virus
isolation
was
carried
out
as
previously
described.
11
All
sequences
of
the
primers/probes
and
PCR
amplification
condition
are
listed
in
Table
S1.
3.3.
Sequencing
and
phylogenetic
analysis
One-step
RT-PCR
was
performed
to
amplify
the
complete
VP1
region
of
EV71
and
CA16
as
previously
described.
11,18
PCR
ampli-
cons
were
sent
to
Invitrogen
BioTechnologies
(Shanghai,
China)
for
DNA
sequencing
using
an
automated
ABI
3730
DNA
sequencer.
The
sequences
obtained
in
this
study
were
screened
using
online
BLAST
Search
tool
(http://blast.ncbi.nlm.nih.gov/Blast.cgi)
to
search
for
the
most
similar
sequences.
Nucleotide
sequences
were
aligned
with
reference
sequences
using
Clustal
W
program
implemented
in
MEGA
5.0.
Phylogenetic
analyses
were
carried
out
as
previously
described.
19
Bootscan
analyses
on
the
genomes
of
our
EV71
and
CA16
isolates
were
conducted
as
previously
described.
20
3.4.
Detection
of
neutralizing
antibody
Sera
from
258
healthy
donors
randomly
recruited
from
Cixi
dis-
trict
of
Ningbo
were
collected
in
April,
2011,
and
stored
at
40
C
before
use.
Neutralizing
antibodies
were
detected
as
previously
described
with
some
modifications.
21
Briefly,
after
inactivated
at
56
C
for
30
min,
the
sera
were
2-fold
serially
diluted
and
incu-
bated
with
equal
volume
(50
L)
of
the
EV71
isolate
(C4a),
the
CA16
isolate
(B1)
or
CA16
(genotype
A)
with
a
tissue
culture
infective
dose
(TCID50)
of
100
for
2
h
at
37
C.
Human
rhabdomyosarcoma
cells
(1
×
10
4
)
were
then
added
to
the
serum-virus
suspensions
in
microplates
in
duplicate
and
incubated
at
35
C
for
7
d.
An
antibody
titer
of
1:8
was
considered
positive.
3.5.
Statistical
analysis
Chi-square
test
was
used
to
determine
the
differences
in
etiological
compositions
and
the
seropositivities
of
neutralizing
antibodies.
We
also
evaluated
linear
trends
in
the
seropositivities
of
neutralizing
antibodies
to
EVs
with
increasing
age.
The
odds
ratio
(OR)
and
95%
confidence
interval
(CI)
for
the
factors
associated
with
the
severity
of
HFMD
were
calculated
in
univariate
analy-
sis.
The
factors
contributing
independently
to
HFMD
severity
were
determined
using
a
forward
stepwise
multivariate
regression
anal-
ysis
(P
entry
=
0.05,
P
removal
=
0.10).
The
adjusted
ORs
were
estimated
by
the
adjustments
for
appropriate
significant
factors
obtained
in
univariate
analysis.
All
statistical
tests
were
two-sided
and
per-
formed
using
the
Statistical
Program
for
Social
Sciences
(SPSS16.0
for
Windows,
SPSS,
Chicago,
IL).
A
P
value
of
<0.05
was
considered
as
statistically
significant.
4.
Results
4.1.
Distribution
of
HFMD
cases
A
total
of
37,404
HFMD
cases
were
reported
via
NNDRS
from
May
2008
to
December
2011
in
Ningbo.
One
hundred
ninety-
six
of
them
were
classified
as
severe
cases.
Twelve
died
of
the
disease.
Using
official
demographic
data
in
2009–2011,
annual
inci-
dence
of
HFMD
was
144.8/100,000
in
the
entire
population
and
3066.8/100,000
in
the
population
of
5
years
(Table
1).
The
cases
of
5
years
accounted
for
95.6%.
The
peak
appeared
in
the
group
of
1–3
years
old,
accounting
for
72.3%
of
all
HFMD
cases
(Fig.
S1).
The
ratio
of
male
to
female
cases
was
1.59:1.
The
epidemic
affected
all
11
districts;
however,
the
incidence
was
higher
in
Beilun
and
Jiangbei
districts
than
other
districts
and
changed
during
the
years
(Fig.
1).
The
occurrence
of
HFMD
had
an
apparent
seasonal
distribu-
tion
with
the
peak
in
June
(Fig.
2).
The
severe
cases
were
frequently
diagnosed
from
May
to
August.
They
accounted
for
0.83,
3.40,
and
10.68
of
all
HFMD
cases;
whereas
the
fatalities
of
the
severe
cases
were
20.00%,
10.68%,
and
4.25%
in
2009,
2010,
and
2011,
respectively.
4.2.
Etiological
characterization
Clinical
specimens
of
2719
reported
cases
including
196
severe
and
12
fatal
cases
were
examined
in
this
period.
Of
those,
2360
(86.8%)
including
179
severe
and
11
fatal
cases
were
laboratory-
confirmed
cases
whose
specimens
were
positive
for
Pan-EVs
by
Page 2
344 H.
Ni
et
al.
/
Journal
of
Clinical
Virology
54 (2012) 342–
348
Table
1
The
hand,
foot,
and
mouth
disease
cases
reported
via
the
National
Notifiable
Disease
Reporting
System
in
Ningbo,
2008–2011.
Year
Number
of
cases
Sex
ratio
(male:female)
Annual
incidence
rate
(per
100,000)
Ratio
of
0–5
year-old
cases/all
cases
(%)
Number
of
severe
cases
Number
of
fatal
cases
Number
of
home-care
cases
(%)
Whole
population
0–5
years
population
2008
a
4359 1.60
––
95.3
3
0
2896
(66.4)
2009
6021
1.79
79.1
1699.7
96.9
5
1
4192
(69.6)
2010 13,828
1.59
181.8
3841.3
95.4
47
5
8832
(63.9)
2011
13,196
1.51
173.5
3659.5
95.2
141
6
8135
(61.6)
Total
37,404
1.59
144.8
3066.8
95.6
196
12
24055
(64.3)
a
The
cases
were
reported
since
May
2,
2008.
real-time
RT-PCR.
The
positive
rates
of
pathogens
identified
in
the
stool
specimens,
anal
swabs,
and
throat
swabs
were
89.90%,
86.70%,
and
63.50%,
respectively.
The
cases
from
Yuyao,
Yinzhou,
and
Cixi
districts
accounted
for
27.4%,
20.3%,
and
13.7%
of
the
laboratory-
confirmed
cases,
respectively.
Of
the
2360
cases,
the
infection
with
EV71,
CA16,
and
other
EVs
accounted
for
63.7%,
24.0%
and
12.3%,
respectively.
EV71
were
more
frequently
identified
in
fatal
and
severe
cases
than
in
mild
cases
(Table
2).
The
VP1
coding
region
of
EV71
from
78
laboratory-confirmed
cases
(71
stool/anal
swabs
and
7
throat
swabs)
including
7
fatal
and
17
severe
cases
was
sequenced.
Divergence
in
the
nucleotide
acids
was
1.5%
(range:
0–4.8%);
while
divergence
in
the
deduced
amino
acids
was
0.2%
(range:
0–1.7%).
No
severe
or
fatal
case-specific
amino
acid
variations
were
identified.
The
epidemic
strain
in
Ningbo
was
phylogenetically
linked
to
clade
C4a
(Fig.S2),
the
same
strain
identified
in
Shandong
in
2007.
11
The
VP1
of
CA16
from
21
mild
cases
(19
stool/anal
swabs
and
2
throat
swabs)
was
sequenced.
Divergence
in
the
nucleotide
acids
was
5.3%
(range:
0–9.2%);
while
divergence
in
the
deduced
amino
acids
was
0.1%
(range:
0–0.3%).
Phylogenesis
indicated
that
CA16
isolated
in
Ningbo
belonged
to
subgroup
B1
(Fig.S3).
We
sequenced
the
full-length
genomes
of
EV71
viruses
iso-
lated
from
8
laboratory-confirmed
cases,
including
3
fatal
and
2
severe
cases,
and
those
of
CA16
viruses
isolated
from
2
mild
cases.
The
genomes
of
EV71
from
fatal
and
non-fatal
cases
were
nearly
identical.
The
full-length
sequence
of
EV71
shared
the
homology
of
98.0%
(range:
97.2-99.4%)
in
nucleotide
and
99.5%
(range:
99.2–99.8%)
in
amino
acid
within
the
8
isolates.
The
full-
length
sequences
of
the
CA16
isolates
shared
the
homology
of
75.2%
(range:
75.1-75.3%)
in
nucleotide
and
87.6%
(range:
87.3-
87.8%)
in
amino
acid
with
the
EV71
isolates.
Our
EV71
isolate
(GenBank:
JQ086366)
showed
high
sequence
similarity
(average
95.7%)
to
EV71/Guangdong 2009/JF799986,
a
C4
strain
isolated
in
Guangdong,
2009,
before
nt.4135
in
the
2C
noncapsid
protein-
coding
region;
however,
a
higher
similarity
(average
90.3%)
to
EV71/SHZH98/AF302996,
a
strain
isolated
in
Shenzhen,
Guangdong
province,
1998,
after
nt.4136
was
observed
(Fig.S4).
Our
CA16
B1a
(GenBank:
JQ354992)
isolates
showed
higher
similarities
to
CA4
Fig.
1.
The
geographic
distribution
of
hand,
foot,
and
mouth
disease
(HFMD)
patients
in
Ningbo,
Zhejiang
Province,
China
from
2009
to
2011.
The
epidemic
covered
all
11
districts
of
Ningbo;
however,
the
cases
were
concentrated
in
Beilun,
Jiangbei,
Haishu,
Jiangdong
and
Yinzhou,
the
central
districts
of
Ningbo.
The
data
in
2008
was
not
included
because
of
the
missing
data
before
May
2,
2008.
Page 3
H.
Ni
et
al.
/
Journal
of
Clinical
Virology
54 (2012) 342–
348 345
Time (Month)
Number of HFMD (Cases)
Fig.
2.
Temporal
distribution
of
hand,
foot,
and
mouth
disease
(HFMD)
patients
in
Nignbo,
Zhejiang
Province,
China,
from
2010
to
2011.
The
number
of
HFMD
patients
had
an
apparent
seasonal
distribution
with
the
peak
in
June.
(GenBank:
AY421762)
at
the
5
UTR
region
and
EV71
prototype
strain
BrCr
after
nt.3683
in
the
2A
noncapsid
protein-coding
region
(Fig.S5).
Online
BLAST
search
indicated
that
the
VP4
sequences
from
38
non-EV71,
non-CA16
EVs
from
the
randomly
selected
cases
in
2008–2010
were
mostly
similar
to
those
of
CA6
(13
cases),
CA10
(7
cases),
CA12
(5
cases),
echo
9
(4
cases),
CA2
(2
cases),
CA3
(2
cases),
CA20
(2
cases),
CA4
(1
case),
CB1
(1
case),
and
echo3
(1
case).
The
sequence
similarities
were
confirmed
by
phylogenetic
analy-
sis
(Fig.
3).
Of
40
non-EV71
and
non-CA16
EVs
from
the
randomly
selected
cases
in
2011,
CA6,
CA10,
and
CA12
accounted
for
30.0%,
25.0%,
and
17.5%,
respectively.
4.3.
Neutralizing
antibodies
to
EV71
and/or
CA16
We
stratified
the
healthy
subjects
into
7
age
groups.
The
posi-
tive
rates
of
neutralizing
antibodies
to
each
of
the
EV71
epidemic
in
Ningbo
and
CA16
A
(GenBank:
JQ315094)
and
both
viruses
increased
with
age;
however,
neutralizing
antibodies
to
two
CA16
B1
isolates
(GenBank:
JQ315095,
JQ315100)
were
all
negative
(Table
3).
4.4.
Differences
in
etiological,
epidemiological
and
clinical
parameters
between
severe
cases
and
mild
cases
Of
the
laboratory-confirmed
cases,
190
severe
and
fatal
cases
and
506
mild
cases
were
individually
interviewed.
A
total
of
538
cases
provided
intact
information
and
were
involved
in
this
assay.
It
was
found
that
infection
with
EV71,
home
care,
exposure
history,
clinical
feature/manifestations
prior
to
the
occurrence
of
typical
neurologic
complications
including
fever,
vomiting,
limb
exanthema,
and
peripheral
neutrophil
ratio
were
significantly
associated
with
an
increased
risk
of
severe
HFMD
(Table
4).
5.
Discussion
HFMD
epidemic
in
Ningbo
is
a
serious
public
health
prob-
lem
although
some
prophylaxis
methods
have
been
implicated
in
schooling
children
since
May
2008.
In
this
study,
64.3%
(24,055/37,404)
reported
HFMD
cases
were
home-care
children
(Table
1).
This
was
possibly
because
the
majority
of
the
reported
cases
were
preschool
children
of
1–3
years
old.
A
study
carried
out
in
Taiwan
indicated
that
household
contacts
contributed
to
52%
of
the
overall
EV71
transmission.
22
Thus,
public
health
inter-
vention
to
household
contacts
should
be
effective
to
interrupt
the
transmission.
The
incidence
in
the
children
<1
year
was
lower
than
that
of
the
1–3
years
groups
(Fig.S1),
possibly
because
maternally
transmitted
neutralizing
antibodies
played
a
protective
role.
23
The
incidences
inversely
related
to
the
positive
rates
of
neutralization
antibody
to
EV71
(Table
3).
This
partially
explains
the
epidemic
pattern
of
HFMD.
The
administration
of
intravenous
immunoglob-
ulin
may
be
effective
for
the
treatment
of
severe
cases.
24,25
The
proportion
of
severe
cases
in
all
HFMD
cases
was
increasing
while
the
fatalities
in
the
severe
cases
were
decreasing
from
2009
to
2011,
possibly
because
immunoglobulin
was
often
used
to
treat
the
severe
cases
(unpublished
data).
Of
the
laboratory-confirmed
cases,
61.3%
were
from
Yuyao,
Yinzhou,
and
Cixi
districts.
Our
sentinel
hospitals
are
mostly
set
there
because
the
three
districts
are
major
land
entries
to
central
Ningbo.
Their
etiological
data
may
partially
explain
the
district
ori-
gin
of
EVs
epidemic
in
central
Ningbo.
Overall,
EV71
C4a
is
the
major
cause
and
frequently
associated
with
HFMD
severity
(Table
2).
CA16
B1
is
another
most
frequently
identified
EVs.
CA6,
CA10,
CA12,
echo
9,
CA2,
CA3,
CA20,
CA4,
CB1,
and
echo3
are
potential
causes
of
HFMD
in
Ningbo.
Of
those,
CA10
and
CA12
are
becoming
important.
Table
2
Etiological
composition
of
the
laboratory-confirmed
cases
in
Ningbo,
2008–2011.
Characteristics
Laboratory-confirmed
cases
Etiological
composition
EV71
(%)
CA16
(%)
Other
enteroviruses
(%)
Total
2360
1503
(63.7)
567
(24.0)
290
(12.3)
Year
of
isolation
*
2008
427
343
(80.4)
42
(9.8)
42
(9.8)
2009
135
70
(51.9)
43
(31.9)
22
(16.3)
2010
1009
591
(58.6)
348
(34.5)
70
(6.9)
2011
789
499
(63.2)
134
(17.0)
156
(19.8)
Clinical
manifestation
Fatal
11
10
(90.9)
0
(0)
1
(9.1)
Severe
179
144
(80.4)
5
(2.8)
30
(16.8)
Mild
2170
1349
(62.2)
562
(25.9)
259
(11.9)
Season
distribution
Winter
(Dec
to
Feb)
185
105
(56.8)
54
(29.2)
26
(14.1)
Spring
(Mar
to
May)
1053
671
(63.7)
252
(23.9)
130
(12.3)
Summer
(Jun
to
Aug)
1075
701
(65.2)
247
(23.0)
127
(11.8)
Autumn
(Sep
to
Nov)
47
26
(55.3)
14
(29.8)
7
(14.9)
*
Pearson’s
Chi-square
test:
2
=
190.523,
P
<
0.001.
Pearson’s
Chi-square
test:
2
=
52.586,
P
<
0.001.
Pearson’s
Chi-square
test:
2
=
6.431,
P
=
0.377.
Page 4
346 H.
Ni
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/
Journal
of
Clinical
Virology
54 (2012) 342–
348
CA6/GD406/China/2009/HQ005441
101/Ningbo/CHN/2010/JQ344141
089/Ningbo/CHN/2010/JQ344140
088/Ningbo/CHN/2010/JQ344139
142/Ningbo/CHN/2009/JQ344121
135/Ningbo/CHN/2009/JQ344119
140/Ningbo/CHN/2009/JQ344120
105/Ningbo/CHN/2009/JQ344118
CA6/P-1808/Kanagawa/2002/AB09474
232/Ningbo/CHN/2009/JQ344129
CA6/Gdula/AB061447
CA6
309/Yuyao/CHN/2010/JQ344147
CA12/P-2200/Kanagawa/2003/AB126210
353/Ningbo/CHN/2009/JQ344134
315/Yuyao/CHN/2010/JQ344148
CA12
145/Ningbo/CHN/2009/JQ344122
172/Ningbo/CHN/2009/JQ344124
CA3/Olson/AY421761
CA3
CA10/2304/Hyogo/2003/AB282808
252/Ningbo/CHN/2009/JQ344131
107/Ningbo/CHN/2010/JQ344142
084/Ningbo/CHN/2009/JQ344117
139/Ningbo/CHN/2010/JQ344146
053/Ningbo/CHN/2010/JQ344137
CA10/A10-22/JN086593
CA10
CA2/Fleetwood/L28146
CA2/P-2202/Kanagawa/2003/AB126198
265/Ningbo/CHN/2009/JQ344133
CA2
EV71/BrCr/U22521
CA4/High Point/AF117695
026/Ningbo/CHN/2010/JQ344135
CA4
171/Ningbo/CHN/2009/JQ344123
264/Ningbo/CHN/2009/JQ344132
CA20/IH-35/Ab06159
CA20
CB1/P-2199/Kanagawa/2003/AB126211
203/Ningbo/CHN/2009/JQ344127
CB1/Con-5/AB061463
CB1
echo3/Morrisey/AB061467
105/Ningbo/CHN/2010/JQ344149
echo3
echo9/Hill/X84981
187/Ningbo/CHN/2009/JQ344126
128/Ningbo/CHN/2010/JQ344145
237/Ningbo/CHN/2009/JQ344130
027/Ningbo/CHN/2010/JQ344136
echo9/isolate DM/AF524867
echo9
100
100
100
100
100
91
100
100
100
100
100
99
99
84
79
97
88
86
76
97
100
97
91
84
90
100
79
0.05
HFMD patients in Ningbo
Fig.
3.
Phylogenetic
trees
constructed
from
the
full-length
VP4
genes
of
the
enteroviruses
amplified
from
the
hand,
foot,
and
mouth
disease
(HFMD)
patients
who
were
positive
for
the
Pan-EVs
but
negative
for
EV71
and
CA16
by
real-time
RT-PCR.
Sequences
of
this
study
are
in
green
solid
circle.
Table
3
Neutralizing
antibodies
to
EV71
and
CA16
in
the
community-based
healthy
population.
Age
group
(years
old)
Number
of
subjects
Positive
rates
of
neutralizing
antibodies
(%)
EV71
C4a
Ningbo
isolate
alone
*
CA16
A
strain
alone
Both
of
EV71
Ningbo
isolate
and
CA16
A
strain
CA16
B1
isolates
1-
37
13.5
43.2
13.5
0
2-
36
22.2
44.4
19.4
0
3-
37
37.8
59.5
29.7
0
4- 37 62.2
70.3
43.2
0
5-
37
67.6
67.6
48.6
0
10-
37
86.5
78.4
70.3
0
20-
37
83.8
78.4
64.9
0
Total
258
53.5
63.2
41.5
0
*
Trend
in
the
seropositivity
of
neutralizing
antibody
to
EV71
isolate
alone
in
the
7
age
groups;
2
=
44.572,
P
trend
<
0.001.
Trend
in
the
seropositivity
of
neutralizing
antibody
to
CA16
A
alone
in
the
7
age
groups;
2
=
11.502,
P
trend
=
0.001.
Trend
in
the
seropositivity
of
neutralizing
antibodies
to
both
of
EV71
isolate
and
CA16
A
in
the
7
age
groups;
2
=
40.703,
P
trend
<
0.001.
Page 5
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Journal
of
Clinical
Virology
54 (2012) 342–
348 347
Table
4
Univariate
and
multivariates
analyses
of
etiological,
epidemiological
and
clinical
parameters
between
mild
cases
and
severe
cases.
Variables
Mild
cases
Severe
and
fatal
cases
OR
(95%
CI)
Adjusted
OR
(95%
CI)
a
Sex
Male 218 117
Reference
Female 136
67
0.92
(0.64–1.33)
Age
2
years
213
76
Reference
<2
years
141
108
2.15
(1.49–3.09)
Pathogen
Other
enteroviruses 137 30 Reference
EV71 217 154
3.24
(2.08–5.06)
2.43
(1.31–4.53)
Living
areas
Rural
221
78
Reference
Integration
of
urban
and
rural
54
68
3.57
(2.30–5.54)
6.50
(3.29–12.86)
Urban
79
38
1.36
(0.86–2.17)
3.57
(1.71–7.46)
Children
care
Schooling
169
35
Reference
Home
care 185
149
3.89
(2.58–5.98)
2.43
(1.31–4.53)
Exposure
to
HFMD
cases
prior–onset
No 316 129 Reference
Yes
38
55
3.55
(2.24–5.62)
10.25
(4.42–23.19)
Tableware
sterilization
Yes
227
59
Reference
No
127
125
3.79
(2.59–5.53)
5.17
(2.91–9.19)
Group
activity
prior–onset
No
247
105
Reference
Yes 107 79 1.74
(1.20–2.51)
2.06
(1.18–3.62)
Fever
<37.5
C
164
18
Reference
37.5
C
190
166
7.96
(4.69–13.51)
8.21
(3.99–16.87)
Limb
exanthema
No 350 166 Reference
Yes
4
18
9.49
(3.16–28.48)
8.91
(1.59–50.10)
Vomiting
No
346
115
Reference
Yes 8
69
25.95
(12.11–55.59)
31.55
(12.40–80.30)
Peripheral
leukocyte
10
×
10
9
/L
87
68
Reference
>10
× 10
9
/L 54 79
1.87
(1.11–2.99)
ND
Neutrophil
ratio
70% 115
79
Reference
>70%
13
39
4.367
(2.19–8.71)
ND
Lymphocyte
ratio
40%
70
60
Reference
>40%
33
45
1.59
(0.90–2.80)
ND
OR,
odds
ratio;
CI,
confidence
interval;
and
HFMD,
hand,
foot,
and
month
disease.
ND,
not
included
in
the
multivariate
analysis
because
of
missing
data
(>40%).
a
Variables
in
the
multivariate
model
were
pathogen,
living
areas,
children
care,
tableware
sterilization,
exposure
to
HFMD
cases
prior–onset,
collective
activity
prior–onset,
fever,
limb
exanthema,
and
vomiting.
The
full-length
genomes
of
EV71
viruses
isolated
from
fatal
and
non-fatal
cases
were
nearly
identical.
Our
data
indicated
that
EV71
C4a
isolate
was
most
likely
a
recombination
of
two
EV71
strains
isolated
in
Guangdong
province,
China
(Fig.S4),
which
is
different
from
other
study.
20
This
difference
is
caused
by
selecting
reference
sequences.
Selection
of
reference
strains
with
high
epidemiologi-
cal
plausibility
is
important
for
the
evolution
and
recombination
analyses.
In
this
study,
we
found
that
the
neutralizing
antibodies
to
the
CA16
subgenotype
B1
isolates
were
negative
in
healthy
subjects
(Table
3).
The
titers
of
neutralizing
antibody
to
EV71
C4a
were
high
whereas
neutralizing
antibody
to
CA16
B1
was
undetectable
at
acute
phase
(unpublished
data).
To
confirm
the
findings,
we
immunized
adult
ICR
mice
and
Wistar
rats
with
the
EV
isolates
following
a
standard
Vero
cell
propagation
and
formalin
inactiva-
tion
protocol.
The
EV71
isolate
generated
neutralizing
antibodies
with
titers
up
to
1:2
14
;
however,
the
CA16
B1
isolate
failed
to
gen-
erate
neutralizing
antibodies
in
both
animal
models
(unpublished
data).
The
reason
could
be
explained
in
two
aspects.
Firstly,
the
antigenicity
of
CA16
B1
might
be
weak
in
nature.
Secondly,
CA16
B1
might
contain
mutations
in
the
antigenic
epitope-encoding
region
and
the
mutants
have
distinct
geographic
distribution.
Zhu
et
al.
21
examined
the
prevalence
of
neutralizing
antibodies
to
CA16B1b
strain
in
China
and
found
geometric
mean
titers
of
the
antibodies
were
geographically
different.
This
challenges
future
immuno-prophylaxis.
We
compared
etiological,
epidemiological
and
clinical
char-
acteristics
between
severe
cases
and
mild
cases.
EV71,
exposure
history,
clinical
feature/manifestations
including
fever,
vomiting,
and
limb
exanthema
prior
to
the
occurrence
of
typical
neurological
complications
were
significantly
associated
with
an
increased
risk
of
severe
HFMD.
These
data
are
helpful
in
early
identification
of
the
HFMD
patients
who
will
develop
into
severe
cases
and
need
a
prompt
and
special
treatment
such
as
intravenous
transfusion
with
immunoglobulin.
Conclusively,
HFMD
mainly
caused
by
EV71
or
CA16
is
still
a
serious
public
health
problem
in
Ningbo.
Public
health
interven-
tion
to
household
transmission
and
immuno-prophylaxis
of
EV71
should
be
important
strategies
in
controlling
the
epidemic.
EV71,
exposure
history,
clinical
feature/manifestations
including
fever,
vomiting,
and
limb
exanthema
are
important
in
early
identification
of
those
who
will
develop
into
severe
cases.
Page 6
348 H.
Ni
et
al.
/
Journal
of
Clinical
Virology
54 (2012) 342–
348
Funding
This
study
was
supported
by
a
key
project
(AWS11L009)
from
Chinese
Military
Logistics,
China;
the
outstanding
young
scholar
fund
(81025015)
from
National
Natural
Science
Foundation
of
China;
a
grant
(2009A190)
from
Zhejiang
Medical
Foundation,
Zhe-
jiang
Province,
China;
and
a
key
project
(2009C50008)
from
Bureau
of
Ningbo
Scientific
and
Technology,
Ningbo,
China.
Competing
interests
None
declared.
Ethical
approval
A
written
informed
consent
was
obtained
from
a
guardian
of
each
patient
who
provided
clinical
specimens
and/or
epidemi-
ological
information.
The
study
protocol
was
approved
by
the
institutional
review
board
of
Ningbo
Center
of
Disease
Control
and
Prevention
(NBCDC200800010).
Appendix
A.
Supplementary
data
Supplementary
data
associated
with
this
article
can
be
found,
in
the
online
version,
at
http://dx.doi.org/10.1016/j.jcv.2012.04.021.
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Page 7
  • Source
    • "Nucleotide sequences were aligned with the above similar sequences and sequences of related viruses [1,2] using Clustal W program implemented in MEGA 6.2. Phylogenetic analyses were carried out as previously described [16]. "
    [Show abstract] [Hide abstract] ABSTRACT: Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne, novel bunyavirus-caused emerging infectious disease. It becomes a threat to public health due to its high fatality rate. To clarify the epidemiological characteristics of SFTS and natural host(s) of SFTS-causing virus (SFTSV) in East China. Serum antibody against SFTSV in 496 healthy villagers was examined by ELISA. SFTSV in acute sera of SFTS cases and lung tissues of house and field mouse/rats were identified using quantitative RT-PCR, cell culture, and sequencing. A total of 22 laboratory-confirmed SFTS cases were diagnosed between 2012 and 2014, of which 5 (22.7%) reported a history of tick-bites. The seroprevalance of SFTSV antibody was 10.5% in healthy villagers. SFTSV genomic RNAs were identified in 2 of 8 Apodemus agrarius but not in Rattus norvegicus (n=40) and Rattus losea (n=4). The 3 segments of SFTSV from 11 SFTS cases and 2 A. agrarius were successfully sequenced. Phylogenetic analyses indicated that at least 3 different SFTSV strains (inland-type, Ningbo-native-type, and their reassortant-type) were present in Ningbo. The 3 segments of the 2 SFTSV isolates from A. agrarius shared great sequence homologies to those isolated from the patients living in nearby villages. The present study indicated that A. agrarius might be a natural host of SFTSV in East China. Copyright © 2015 Elsevier B.V. All rights reserved.
    Full-text · Article · Aug 2015 · Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology
  • Source
    • "In a conventional format [9], [10], [11], [12], [13], [14], [15], the requirement for a post-PCR step (usually an agarose gel electrophoresis) is associated with a high risk of contamination that can seriously jeopardise diagnosis. More recently, the real-time PCR format [16], [17], [18], [19], [20], [21], [22], [23] allows a rapid, specific and sensitive diagnosis with a low risk of contamination. Real-time PCR techniques have been developed in institutions for, and tested almost exclusively on, strains in the populatjons served by these institutions. "
    [Show abstract] [Hide abstract] ABSTRACT: Enterovirus A71 (EV-A71) has recently become an important public health threat, especially in South-East Asia, where it has caused massive outbreaks of Hand, Foot and Mouth disease every year, resulting in significant mortality. Rapid detection of EV-A71 early in outbreaks would facilitate implementation of prevention and control measures to limit spread. Real-time RT-PCR is the technique of choice for the rapid diagnosis of EV-A71 infection and several systems have been developed to detect circulating strains. Although eight genogroups have been described globally, none of these PCR techniques detect all eight. We describe, for the first time, a SYBR Green real-time RT-PCR system validated to detect all 8 EV-A71 genogroups. This tool could permit the early detection and shift in genogroup circulation and the standardization of HFMD virological diagnosis, facilitating networking of laboratories working on EV-A71 in different regions.
    Full-text · Article · Mar 2014 · PLoS ONE
  • Source
    • "Viral RNA was extracted from clinical specimens and viral cultures using a QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. All RNA samples were examined by real time reverse-transcription PCR (rt RT-PCR) using a set of Pan-EV (EV universal primer) probe and primers; positive samples were tested by rt RT-PCR for EV71 and CA16 using specific primers and probes [17]. Rt RT-PCR was performed using AgPath-ID™ One-Step RT-PCR reagents (Applied Biosystems, Foster, CA, USA). "
    [Show abstract] [Hide abstract] ABSTRACT: To analyze the epidemiological characteristics and pathogenic molecular characteristics of an hand, foot, and mouth disease (HFMD) outbreak caused by enterovirus 71 in Linyi City, Shandong Province, China during November 30 to December 28, 2010. One hundred and seventy three stool specimens and 40 throat samples were collected from 173 hospitalized cases. Epidemiologic and clinical investigations, laboratory testing, and genetic analyses were performed to identify the causal pathogen of the outbreak. Among the 173 cases reported in December 2010, the male-female ratio was 1.88: 1; 23 cases (13.3%) were severe. The majority of patients were children aged < 5 years (95.4%). Some patients developed respiratory symptoms including runny nose (38.2%), cough (20.2%), and sore throat (14.5%). One hundred and thirty eight EV71 positive cases were identified based on real time reverse-transcription PCR detection and 107 isolates were sequenced with the VP1 region. Phylogenetic analysis of full-length VP1 sequences of 107 Linyi EV71 isolates showed that they belonged to the C4a cluster of the C4 subgenotype and were divided into 3 lineages (Lineage I, II and III). The two amino acid substitutions (Gly and Gln for Glu) at position 145 within the VP1 region are more likely to appear in EV71 isolates from severe cases (52.2%) than those recovered from mild cases (8.3%). This outbreak of HMFD was caused by EV71 in an atypical winter. EV71 strains associated with this outbreak represented three separate chains of transmission. Substitution at amino acid position 145 of the VP1 region of EV71 might be an important virulence marker for severe cases. These findings suggest that continued surveillance for EV71 variants has the potential to greatly impact HFMD prevention and control.
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