MAJOR ARTICLE

Dengue Hemorrhagic Fever in Infants: A Study

of Clinical and Cytokine Profiles
Nguyen Thanh Hung,1 Huan-Yao Lei,4 Nguyen Trong Lan,1 Yee-Shin Lin,4 Kao-Jean Huang,4 Le Bich Lien,1
Chiou-Feng Lin,4 Trai-Ming Yeh,5 Do Quang Ha,2 Vu Thi Que Huong,2 Lien-Cheng Chen,5 Jyh-Hsiung Huang,7
Lam Thi My,3 Ching-Chuan Liu,6 and Scott B. Halstead8
1
Department of Dengue Hemorrhagic Fever, Childrens Hospital No. 1Ho Chi Minh City, 2Arbovirus Laboratory, Pasteur InstituteHo Chi Minh
City, and 3Department of Pediatrics, University of Medicine and PharmacyHo Chi Minh City, Ho Chi Minh City, Vietnam; Departments of
4
Microbiology and Immunology, 5Medical Technology, and 6Pediatrics, College of Medicine, National Cheng Kung University, Tainan, and 7Division
of Research and Diagnosis, Center for Disease Control, Department of Health, Taipei, Taiwan, Republic of China; 8Uniformed Services University
of the Health Sciences, Bethesda, Maryland

A prospective study of clinical and cytokine profiles of 107 infants with dengue hemorrhagic fever (DHF)/
dengue shock syndrome (DSS) was conducted. Fever, petechiae on the skin, and hepatomegaly were the most
common clinical findings associated with DHF/DSS in infants. DSS occurred in 20.5% of the patients. Hemo-
concentration and thrombocytopenia were observed in 91.5% and 92.5% of the patients, respectively. Serologic
testing revealed that almost all of the patients (95.3%) had primary dengue virus infections. These data
demonstrate that clinical and laboratory findings of DHF/DSS in infants are compatible with the World Health
Organizations clinical diagnostic criteria for pediatric DHF. The present study is the first to report evidence
of production of cytokines in infants with DHF/DSS and to describe the difference between the cytokine
profile of infants with primary dengue virus infections and children with secondary infections. Overproduction
of both proinflammatory cytokines (interferon-g and tumor necrosis factora) and anti-inflammatory cyto-
kines (interleukin-10 and -6) may play a role in the pathogenesis of DHF/DSS in infants.

Dengue viruses are members of the family Flaviviridae
and are of 4 serotypes (DEN-1, DEN-2, DEN-3, and
DEN-4). Dengue virus infections may be asymptomatic
or may lead to undifferentiated fever; dengue fever (DF)
or dengue hemorrhagic fever (DHF) with capillary leak-
age that may progress to hypovolemic shock; or dengue
shock syndrome (DSS), which may be fatal if it is not
treated correctly [1, 2]. Dengue virus infections are se-
rious public health problems in many regions of the
world. More than 2.5 billion people in 1100 countries
worldwide are now at risk for dengue virus infection.
An estimated 50 million infections occur annually, in-
cluding 500,000 cases of DHF/DSS, and at least 2.5%
Received 1 April 2003; accepted 16 July 2003; electronically published 9 January
2004.
Financial support: partial support from National Health Research Institutes, Taiwan
(grant NHRI-CN-CL-8901P).
Reprints or correspondence: Dr. Nguyen Thanh Hung, Dept. of Dengue Hemorrhagic
Fever, Childrens Hospital No. 1Ho Chi Minh City, 341 Su Van Hanh St., District 10,
Ho Chi Minh City, Vietnam (hungdhf@hcm.fpt.vn).
The Journal of Infectious Diseases 2004; 189:22132
 2004 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2004/18902-0008$15.00
of individuals with DHF/DSS die. The vast majority of
cases, nearly 95%, occur in children !15 years of age
[3], and 5% of all DHF/DSS cases occur in infants.
However, since DHF/DSS was reported to occur in in-
fants during their first dengue infections by Halstead
et al. [4] in 1969, only a few research studies, with rather
small numbers of patients, have been published on this
subject [59]. Technical difficulties in obtaining the
rather large blood samples required by research pro-
tocols from small subjects and possible constraints im-
posed by human experimentation protocols may par-
tially explain why DHF/DSS in infants has not been
more comprehensively studied, as has DHF/DSS in older
children [10].
A frequently cited model of the immunopathogen-
esis of DHF/DSS focuses on secondary dengue virus in-
fections in children 11 year of age [1113]. In this model,
aberrant or memory immune activation directed against
dengue virusinfected cells initiates cytokine release, in-
creased vascular permeability, and activation of the blood
clotting system, the hallmarks of DHF/DSS [13]. Com-
parable immunologic studies of the features of se-

Dengue Hemorrhagic Fever in Infants JID 2004:189 (15 January) 221

vere dengue in infants during their first dengue virus infection
have not been published. The research challenge presented by
infant DHF/DSS has been described recently by Halstead et al.
[10]. Studies involving infants with DHF/DSS accompanying
primary dengue virus infections should provide powerful new
evidence with regard to the immune mechanisms underlying
all cases of DHF [14]. In response to the challenge of studying
this unique pathogenetic group, we conducted a prospective
study of clinical and cytokine profiles in infants. In the present
study, we demonstrate that clinical and laboratory findings as-
sociated with DHF/DSS in infants are compatible with the clin-
ical diagnostic criteria for DHF in children of the World Health
Organization (WHO). We also report evidence of cytokine
overproduction in infants with DHF/DSS.
PATIENTS, MATERIALS, AND METHODS
Patients
One hundred eighteen infants !12 months of age who were
admitted to the Department of Dengue Hemorrhagic Fever at
Childrens Hospital No. 1Ho Chi Minh City (Ho Chi Minh
City, Vietnam) between January 1998 and March 2002 with a
clinical diagnosis of suspected DHF according to the criteria
of the WHO [1] were enrolled in the study, provided that a
parent or guardian gave informed consent. According to WHO
criteria, which are based on information derived from children,
patients were classified as having nonshock DHF (grades I and
II) or DSS (grades III and IV) [1].
Patients received routine care from 1 of the authors. For
each patient, basic demographic data, medical history, physi-
cal examination findings, and subsequent progress were re-
corded on a standard data form. Informed consent was ob-
tained from all parents or guardians. The study was approved
by the Scientific and Ethical Committee of Childrens Hospital
No. 1Ho Chi Minh City.
Sample Collection
Paired blood samples were obtained from each patient in the
study, 1 during the acute phase and 1 during the convalescent
phase. Acute-phase blood samples (23 mL) were obtained at
admission to the hospital (day 3 to day 7 after the onset of fever).
Convalescent-phase blood samples (23 mL) were obtained dur-
ing the convalescent phase of the disease (day 8 to day 19 after
the onset of fever). A blood sample from each of 99 mothers of
these patients was obtained at the time of the infants hospital
admission. Serum was separated as quickly as possible, and serum
samples were stored at 70C until they were used.
Laboratory Methods
Serologic testing. Dengue virus infections in infants were
confirmed by viral envelope and membrane (E/M)specific
222 JID 2004:189 (15 January) Hung et al.
capture IgM and IgG ELISAs, using dengue virus and Japanese
encephalitis virus (JEV) as antigens, at the Center for Disease
Control, Department of Health, Taipei, Taiwan. Dual analyses
of E/M-specific capture IgM and IgG ELISAs and NS1 serotype
specific IgG ELISAs were used to differentiate between primary
and secondary dengue virus infections, as described elsewhere
[15]. Serum samples from the mothers were tested to determine
their immune status to dengue virus by use of NS1 serotype
specific IgG ELISAs.
E/M-specific capture IgM and IgG ELISAs in which diluted
pooled viral antigens from culture supernatants of DEN-1,
DEN-2, DEN-3, DEN-4, and JEV-infected Vero cells were
used as antigens were performed to measure levels of IgM and
IgG antibodies in paired serum samples from the infants. The
enzyme activity was finally developed, and the optical density
(OD) was determined 30 min later at the dual wavelengths of
405 and 630 nm with a Dynatech MR700 microplate reader [15].
NS1 serotypespecific IgG ELISAs in which diluted NS1-
containing culture supernatants of DEN-1, DEN-2, DEN-3,
DEN-4, and JEV-infected Vero cells were used as antigens were
performed to measure levels of NS1-specific IgG antibody in
serum samples from infants and mothers. The enzyme activity
was developed, and ODs were determined [15].
The ODs of culture supernatants of Vero cells with and with-
out dengue virus infection were designated the test absorbance
and negative control value, respectively, for each sample in the
ELISA. Positivity was determined by comparison to individual
negative controls. A positive sample was a sample with a ratio
of test absorbance to negative control of 2.0, and a negative
sample was a sample with a ratio of !2.0. For serum samples
with positive results of capture IgM and IgG ELISAs, a ratio
of IgM to IgG of 1.2 was used to identify a primary dengue
virus infection, and a ratio of !1.2 was used to identify a sec-
ondary dengue virus infection. For serum samples with positive
NS1-specific IgG antibody responses, NS1 serotype was deter-
mined by the ratio of the highest OD value to the second highest
OD value read from the 4 dengue virus serotypes. Positive
serotype specificity was defined by an OD ratio 1.2, and neg-
ative serotype specificity was defined by an OD ratio !1.2. NS1
serotypespecific IgG ELISA was used to identify primary den-
gue virus infection; primary infection was defined by (1) a
negative NS1-specific IgG antibody response for serum samples
obtained between days 1 and 14 of illness or (2) a positive
serotype specificity for serum samples obtained after 9 days
of illness. Secondary dengue virus infection was defined by (1)
a positive NS1-specific IgG antibody response for serum sam-
ples obtained between days 1 and 8 of illness or (2) a positive
NS1-specific IgG antibody response and negative serotype spec-
ificity at any time after the onset of symptoms [15].
Cytokine assays. The plasma levels of 6 cytokines (interferon
[IFN]g, tumor necrosis factor [TNF]a, interleukin [IL]10,
IL-6, IL-4, and IL-2) of the patients were measured simulta-
neously by the BD Human Th1/Th2 Cytokine Cytometric Bead
Array Kit-II (BD Biosciences Pharmigen) in a 50-mL sample,
according to the manufacturers instructions. The detection limits
of the kit for IFN-g, TNF-a, IL-10, IL-6, IL-4, and IL-2 were
7.1, 2.8, 2.8, 3.0, 2.6, and 2.6 pg/mL, respectively.
Protein C and S assays. Plasma levels of proteins C and
S were measured by use of commercial ELISA kits (Helena
Laboratories). The laboratory methods used and the interpre-
tation of results for all kits followed the manufacturers in-
structions. Determinations of cytokine and protein C and S
levels in the patients samples were performed at the Depart-
ments of Microbiology and Immunology and of Medical Tech-
nology, Medical College, National Cheng Kung University, Tai-
nan, Taiwan.
Complete blood counts, coagulation tests, liver and renal
function tests, and ionograms. Complete blood counts (Care-
side H-2000 counter), coagulation tests (Diagnostica Stago), liver
and renal function tests, and ionograms (R5A 800A kit; Hycel
Groupe Lisabio) were performed at the hospital laboratory.
Data and Statistical Analysis
The statistical significance of differences in normally distributed
data was compared using analysis of variance; the Kruskal-Wallis
test for 2 groups was used when the variances in the samples
differed. Statistical analyses were performed with EpiInfo 2000,
version 1.1 (Centers for Disease Control and Prevention). P !
.05 was considered to be statistically significant.
RESULTS
Clinical findings. On the basis of E/M-specific capture IgM
and IgG ELISAs using dengue virus and JEV as antigens, dengue
virus infection was confirmed to be the cause of illness for 107
of 118 infants hospitalized with DHF. Dengue virusspecific
IgM and/or IgG antibodies with limited cross-reactivity to JEV
were found in serum samples from these 107 infants (data not
shown). The limited cross-reactivity was most likely the result
of the naive status of immune memory to other flavivirus of
these infants. Eighty-five of these 107 infants were categorized
as having nonshock DHF (grade I, 1 infant; grade II, 84 infants),
and 22 were categorized as having DSS (grade III, 17 infants;
grade IV, 5 infants). The age distribution is shown in figure 1.
Some cases were observed in infants !3 months of age, but the
largest numbers of cases were in infants who were 69 months
of age (mean, 6.7 months; range, 111 months). The mean age
of infants with DSS was significantly higher than that of infants
with nonshock DHF (8.2 vs. 6.4 months; P p .002). Twenty
(90%) of 22 DSS patients were infants 6 months. The male-
to-female ratio was 57:50 (1.1:1).
Table 1 shows the clinical characteristics, laboratory findings,
Dengue Hemorrhagic Fever in Infants JID 2004:189 (15 January) 223
Figure 1. Age distribution, by month, of 107 infants with dengue hem-
orrhagic fever (DHF)/dengue shock syndrome (DSS). Nos. of infants are shown
above columns. Few cases were observed in infants !3 months of age; most
cases occurred in infants between the ages of 6 and 9 months. White columns,
All infants with DHF or DSS; black columns, infants with DSS.
and treatment for the patients. All patients had high, contin-
uous fever that lasted 213 days (mean, 5.2 days). Almost all
patients had petechiae on the skin (106 [99.0%] of 107). Eight
patients (7.4%) had gastrointestinal (GI) bleeding (hematem-
esis or melena). Surprisingly, epistaxis and gum bleeding were
not recorded for any patient. Hepatomegaly was detected in
104 patients (97.1%). Liver size ranged from 1 to 6 cm below
the right costal margin. Interestingly, infants with DSS had a
much greater degree of liver enlargement than did those with
nonshock DHF (mean, 3.8 vs. 2.8 cm; P p .000). Splenomegaly
was detected in 7 patients (6.5%). DSS was observed in 22
patients (20.5%). Shock occurred on day 3 to day 6 after the
onset of fever (mean, 4.6 days). Thirteen (59.0%) of these 22
patients still had fever at the onset of shock; fever lasted from
1 to 4 days after onset of shock (mean, 1.8 days). Ten (9.3%)
of the 107 patients had neurologic signs (dengue encephalop-
athy) manifested by convulsion (7 patients), lethargy (4 pa-
tients), and coma (4 patients). In addition, nonspecific signs
and symptoms (cough, runny nose, and diarrhea) were noted
in 46 patients (42.9%), 29 patients (27.1%), and 20 patients
(18.6%), respectively. Coinfections seen in infants with DHF/
DSS in the present study were pneumonia (4 patients), bron-
chitis/bronchiolitis (4 patients), and shigellosis (2 patients).
Laboratory findings. Hemoconcentration, manifested by
an increase in hematocrit (Hct) of 20% above the convales-
cent-phase value, was observed in 98 patients (91.5%). In the
other 9 patients (8.4%), Hct increased by 10%19%. The
peak Hct, as well as increase in Hct, for patients with DSS was
significantly higher than that for patients with nonshock DHF
(mean, 42.6% vs. 39.1% [P p .000] and 41.9% vs. 32.5%
[P p .003], respectively). Thrombocytopenia was found in 99
Table 1. Clinical characteristics, laboratory findings, and treatments for 85 infants with nonshock dengue hemorrhagic fever (DHF)
and 22 infants with dengue shock syndrome (DSS).

Infants with
a
Variable All infants nonshock DHF Infants with DSS P

Clinical characteristic
Age, mean months  SD (range) 6.7  2.5 (111) 6.4  2.5 (111) 8.2  2.0 (411) .002
Duration of fever, mean days  SD (range) 5.2  1.7 (213) 5.1  1.8 (213) 5.6  1.4 (49) .09
Bleeding manifestations, no. (%) of infants
Petechiae 106 (99.0) 84 (98.8) 22 (100)
Gastrointestinal bleeding 8 (7.4) 5 (5.8) 3 (13.6)
b
Liver size, mean cm  SD (range) 3.0  1.0 (16) 2.8  0.9 (16) 3.8  1.1 (26) .000
Laboratory findings
Peak hematocrit, mean %  SD (range) 39.8  4.4 (3060) 39.1  4.0 (3060) 42.6  4.7 (3554) .000
Increase in hematocrit, mean %  SD (range) 34.4  12.7 (1060) 32.5  12.0 (1057) 41.9  12.7 (2160) .003
Lowest platelet count, mean cells 103/mm3  SD (range) 61.9  34.8 (40190) 65.3  36.3 (40190) 49.0  24.9 (20100) .02
AST level, mean U/L  SD (range) 960.2  1977.7 (337890) 2307.8  3353.9 (547890) 421.2  585.9 (332183) .1
ALT level, mean U/L  SD (range) 314.1  517.3 (212190) 583.3  799.4 (492190) 206.5  319.1 (211377) .3
PT, mean s  SD (range) 27.2  28.5 (10.499) 12.2  1.4 (10.414.5) 40.1  34.7 (13.299) .008
APTT, mean s  SD (range) 80.1  36.5 (32.6120) 55.6  32.1 (32.6120) 101.1  26.5 (59.4120) .01
Fibrinogen level, mean g/L  SD (range) 1.2  0.6 (0.62.7) 1.7  0.6 (1.12.7) 0.7  0.2 (0.61.1) .002
Protein C
c
Acute-phase samples, mean %  SD (range) 54.5  19.6 (6.394.4) 56.7  15.5 (28.384.3) 49.8  26.1 (6.394.4) .3
c
Convalescent-phase samples, mean %  SD (range) 70.1  14.8 (48.9105.9) 68.9  11.6 (50.390.3) 72.8  21.6 (48.9105.9) .7
Protein S
c
Acute-phase samples, mean %  SD (range) 55.4  21.1 (1.383.7) 57.7  17.3 (1.387.2) 50.6  27.5 (3.383.7) .6
c
Convalescent-phase samples, mean %  SD (range) 82.5  17.5 (42.3120) 81.1  16.4 (42.3103.7) 85.7  20.6 (58.1120) .8
Serologic response, no. (%) of infants
Primary infection 102 (95.3) 81 (75.7) 21 (19.6)
Secondary infection 5 (4.6) 4 (3.7) 1 (0.9)
Treatment
Average amount of iv fluid, mean mL/kg  SD (range) 104.3  29.4 (27.5211.7) 102.5  29.7 (27.5211.7) 111.0  28.1 (52178.5) .1
Average amount of dextran 40, mean mL/kg  SD (range) 53.6  25.4 (28100) 35.5  7.5 (2850) 63.3  26.5 (28100) .01
Duration of iv fluid administration, mean h  SD (range) 24.9  8.0 (653) 25.7  7.9 (953) 22.0  7.8 (641) .9

NOTE. ALT, alanine aminotransferase; APTT, activated partial thromboplastin time; AST, aspartate aminotransferase; iv, intravenous; PT, prothrombin time.
a
Nonshock DHF group vs. DSS group; calculated using the Kruskal-Wallis test.
b
Below right costal margin.
c
For infants with DHF/DSS vs. control infants, P p .000 (Kruskal-Wallis test). Mean plasma levels  SD of proteins C and S in the 10 control infants were
105.0%  17.4% (range, 84.6%143.3%) and 110.9%  9.7% (range, 96.6%126.7%), respectively.

patients (92.5%). The lowest platelet count for infants with DSS
was significantly lower than that for infants with nonshock DHF
(P p .02; table 1). The lowest platelet count was significantly
lower for infants with GI bleeding than for infants without
(mean, 39.6 10 3 vs. 64.1 10 3 cells/mm3; P p .02; table 2).
Liver function tests and ionograms were performed for only
28 and 42 patients, respectively, because of the difficulty of ob-
taining enough blood for many tests from infants. Elevated serum
levels of aspartate aminotransaminase (AST) and alanine ami-
notransaminase (ALT) were seen in 26 patients (92.8%) and 23
patients (82.1%), respectively. There was a significant difference
in serum levels of AST and ALT in infants with and without GI
bleeding (mean, 3739.8 vs. 356 U/L [P p .03] and 1119.4 vs.
139.1 U/L [P p .002], respectively) (table 2). However, serum
levels of AST and ALT did not differ significantly between infants
with nonshock DHF and those with DSS (P p .1 and P p .3,
respectively). Hyponatremia was detected in 23 patients (54.7%),
and hypocalcemia and hypokalemia were detected in 6 patients
(14.2%) and 2 patients (4.7%), respectively.
Dual analyses of E/M-specific capture IgM and IgG ELISA
and NS1 serotypespecific IgG ELISA results showed that 102
patients (95.3%) had primary dengue virus infections and only
5 patients (4.6%) had secondary dengue virus infections.
Ninety-eight (98.9%) of 99 mothers whose serum samples were
available were found to be seropositive for dengue virus by
means of NS1 serotypespecific IgG ELISA. Based on NS1 sero-
type-specific IgG ELISA results, most of those 98 mothers (86
mothers [87.7%]) had experienced 2 dengue virus infections
during their lifetime, whereas only 12 mothers (12.2%) had
previously experienced a single dengue virus infection. Rep-

224 JID 2004:189 (15 January) Hung et al.

Table 2. Comparison between infants with nonshock dengue hemorrhagic fever/dengue shock syndrome who had gastrointestinal
(GI) bleeding and those who did not.

Infants with GI bleeding Infants without GI bleeding

No. for No. for
whom data whom data
Finding were available Mean value  SD (range) were available Mean value  SD (range) P
a
Liver size, cm 8 4.5  1.6 (36) 96 2.9  0.9 (16) .002
Lowest platelet count,
cells 103/mm3 8 39.6  23.8 (2090) 99 64.1  35.5 (14190) .02
Liver function test 5 23
AST level, U/L 3739.8  3572.9 (477890) 356.0  584.5 (332615) .03
ALT level, U/L 1119.4  792.3 (1162190) 139.1  186.1 (21818) .002
Coagulation test 3 10
PT, s 34.9  39.0 (10.480) 24.9  26.8 (11.199) .9
APTT, s 93.5  45.8 (40.5120) 76.1  35.2 (32.6120) .6
Fibrinogen level, g/L 1.3  0.7 (0.62.1) 1.1  0.6 (0.62.7) .6
Proteins C and S in acute-phase samples 3 50
Protein C, % 31.3  24.9 (6.356.1) 35.0  29.2 (3.361) .08
Protein S, % 55.9  18.7 (14.494.4) 56.6  20.3 (1.387.2) .1

NOTE. ALT, alanine aminotransferase; APTT, activated partial thromboplastin time; AST, aspartate aminotransferase; PT, prothrombin time.
a
Below right costal margin.

resentative results of E/M-specific capture IgM and IgG ELISAs
and/or NS1 serotypespecific IgG ELISAs for paired infants and
mothers are presented in table 3. Patients 14 had primary
dengue virus infections (all caused by DEN-3); patient 5 had
secondary dengue virus infection. Positive NS1-specific IgG
antibody responses and negative serotype specificity (NS1 sero-
typing OD ratio !1.2) in serum samples from the mothers of
patients 14 (M6, M26, M52, and M65, respectively) showed
that these mothers had experienced 2 previous dengue virus
infections, and the other mother (M58) had experienced a sin-
gle dengue virus infection (caused by DEN-2), demonstrated
by positive NS1-specific IgG antibody responses and an NS1
serotyping OD ratio 1.2 (table 3).
Chest radiography was performed for 31 patients. Right-side
pleural effusion was demonstrated in 8 patients, bilateral pleural
effusion in 8 patients, bronchitis/bronchiolitis in 4 patients,
and pneumonia in 4 patients.
Treatment and result. One hundred patients (93.4%; 78
patients with nonshock DHF and 22 patients with DSS) re-
ceived intravenous fluid replacement with Ringers lactate (RL)
or 5% dextrose in RL solution (5% dextrose in RL solution is
used to treat patients with nonshock DHF, not patients with
DSS). The mean amount of intravenous fluid was 104.3 mL/
kg (range, 27.5211.7 mL/kg), administered over a mean period
of 24.9 h (range, 653 h). In the present study, there was no
significant difference in the mean amount or in the duration
of intravenous fluid replacement between patients with non-
shock DHF and those with DSS (P p .1 and P p .9, respec-
tively). Administration of colloid solution (dextran 40) was
indicated in 20 patients (18.6%). The mean amount admin-
istered was 53.6 mL/kg (range, 28100 mL/kg); this was cor-
related with severity of the disease (P p .01; table 1). Eight
patients (7.4%), 5 of whom had severe GI bleeding, received
a transfusion of fresh whole blood (FWB; mean amount, 24.7
mL/kg; range, 1371 mL/kg).
With proper case management and good nursing care, almost
all of the patients completely recovered, but 4 patients died;
thus, the case-fatality rate was 3.7% among infants with DHF/
DSS in this study. Of the patients who died, 2 had prolonged
shock, 3 had encephalopathy, 2 had respiratory failure, and 3
had massive GI bleeding. Clinical characteristics and laboratory
findings for these 4 patients are shown in table 4.
Cytokine profile in infants with DHF/DSS. The levels of
IFN-g in the acute-phase samples from infants with DHF/DSS
were significantly higher than those in samples from healthy
control infants (mean, 56.2 vs. 4.1 pg/mL; P p .01; table 5).
When the data are pooled and the kinetic changes in cytokine
blood levels are plotted by day after fever onset, levels of IFN-
g in infants with DHF/DSS are found to have increased on day
4 to day 6 after the onset of fever and to have rapidly decreased
on day 7 and during the convalescent phase. The concentration
of IFN-g in convalescent-phase samples from the patients was
somewhat higher than that for control subjects, but was not
significantly different (mean, 18.5 vs. 4.1 pg/mL; P p .3; figure
2). Similar results were observed when the TNF-a levels in the
acute- and convalescent-phase samples were compared with
those in samples from control subjects. Infants with DHF/DSS
had significantly higher TNF-a levels in acute-phase samples

Dengue Hemorrhagic Fever in Infants JID 2004:189 (15 January) 225

Table 3. Representative results of capture IgM and IgG ELISAs and NS1 serotypespecific IgG ELISAs for samples from paired infants with nonshock dengue hemorrhagic
fever/dengue shock syndrome and samples from mothers.

Days between
Capture IgM and IgG ELISA results NS1 serotypespecific IgG ELISA results
fever onset
and sample Disease Ratio of NS1
Patient, serum sample Sex/age collection grade DEN IgM JEV IgM DEN IgG JEV IgG IgM to IgG JEV DEN-1 DEN-2 DEN-3 DEN-4 CC typing Interpretation

JEPC 0.235 1.501 0.211 1.591 0.67 0.13 0.14 0.15 0.14 0.14
D2PC 2.446 0.143 0.913 0.258 0.13 0.66 1.45 0.26 0.19 0.10 2.20
D1234PC 0.572 0.2 3.719 1.575 0.36 2.73 3.03 3.04 2.95 0.20 1.00
Infants
1 F/10 months III Primary infection (DEN-3)
981807E 6 3.808 0.418 0.219 0.155 17.39 0.29 0.29 0.27 0.27 0.28 0.30
982015E 19 3.783 0.439 3.747 0.182 1.01 0.19 0.25 0.46 1.21 0.29 0.13 2.67
2 M/5 months II Primary infection (DEN-3)
983295E 5 1.443 0.205 0.143 0.149 10.09 0.13 0.13 0.16 0.13 0.12 0.13
983629E 16 3.902 0.349 3.279 0.182 1.19 0.19 0.52 0.34 1.92 0.30 0.13 3.70
3 M/2 months II Primary infection (DEN-3)
991749E 5 0.852 0.222 0.193 0.153 4.41 0.25 0.46 0.40 0.20 0.27 0.15
991856E 15 3.737 0.309 1.077 0.161 3.47 0.22 0.59 0.43 1.20 0.36 0.13 2.04
4 F/2 months II Primary infection (DEN-3)
20001898E 5 3.759 0.241 0.185 0.182 20.32 0.16 0.22 0.18 0.20 0.15 0.14
20002021E 11 3.821 0.34 3.578 0.244 1.07 0.15 0.31 0.19 0.64 0.16 0.11 2.08
5, 20020017E M/7 months 9 III 0.293 0.282 0.709 0.308 0.41 0.16 1.97 2.02 1.70 2.18 0.11 1.08 Secondary infection
Mothers
M6 26 years 0.25 2.41 2.80 2.67 1.98 0.12 1.04 Secondary infection
M26 25 years 0.44 1.50 1.83 1.66 1.20 0.20 1.10 Secondary infection
M52 31 years 0.61 2.85 3.84 2.97 3.59 0.25 1.07 Secondary infection
M65 28 years 0.69 2.74 3.05 2.82 2.44 0.28 1.08 Secondary infection
M58 23 years 0.22 0.27 0.61 0.35 0.40 0.28 1.51 Primary infection

NOTE. JEPC, D2PC, D1234PC, and CC were positive controls for JEV, primary DEN-2 infection, and secondary DEN infection and a negative control, respectively. M6, M26, M52, and M65 were the
mothers of infants 1, 2, 3, and 4, respectively. DEN, dengue virus; JEV, Japanese encephalitis virus.
Table 4. Clinical and laboratory data for 4 infants with fatal dengue shock syndrome.

Patient

Variable 1 2 3 4
Hospital no. 86018/98 115,637/98 120,896/98 191,154/00
Age, months 7 5 11 11
Sex F M F M
Weight, kg 7 7 8.5 10
Date of admission 22 May 1998 13 Jul 1998 24 Jul 1998 29 Nov 2000
Date of death 23 May 1998 14 Jul 1998 28 Jul 1998 30 Nov 2000
Disease grade III III III III
Clinical finding
a
Consciousness Coma Coma Coma Coma
Convulsion No No Yes Yes
Petechiae Yes Yes Yes Yes
GI bleeding Yes Yes No Yes
b
Liver size, cm 6 5 4 4
Jaundice No No No No
Shock Yes Yes Yes Yes
Prolonged shock No No Yes Yes
Respiratory failure No No Yes Yes
Laboratory findings
Hematocrit, min/max % 22/40 22/44 32/44 16/40
Lowest platelet count, 20 30 32 20
cells 103/mm3
AST/ALT level, U/L 7220/2190 7890/1303 174/44 1380/458
Total/conjugated bilirubin, 0.6/0.2 0.8/0.3 0.3/0.1 0.6/0.2
mg/dL
Ionogram result, mmol/L
Na+ 132 138 132 126
K+ 5.1 5.1 5.2 6.9
Ca++ (ionized) 1.1 0.73 1.2 0.88
Coagulation
PT, s 99 99 1120 80
APTT, s 1120 1120 1120 1120
Fibrinogen, g/L !0.6 !0.6 !0.6 !0.6
Cerebrospinal fluid Normal ND ND Normal
ELISA result, IgM/IgG 1.643/0.202 4.01/0.173 1.838/0.125 2.787/0.241
ELISA interpretation Primary infection Primary infection Primary infection Primary infection
Treatment Infusion with RL, dextran 40, Infusion with RL, dextran 40, Infusion with RL, dextran 40, Infusion with RL, dextran 40,
FWB, gelatin FWB,platelet concentrates, dopamine, dobutamine; NCPAP FWB, dopamine; NCPAP
dopamine, dobutamine

NOTE. ALT, alanine aminotransferase; APTT, activated partial thromboplastin time; AST, asparate aminotransferase; FWB, fresh whole blood; GI, gastro-
intestinal; RL, Ringers lactate; NCPAP, nasal continuous positive airway pressure; ND, not determined; PT, prothrombin time.
a
Coma at the terminal stage.
b
Below right costal margin.

than did control subjects (mean, 9 vs. 0.8 pg/mL; P p .01; table
5). TNF-a levels were elevated on day 4 to day 7 after the onset
of fever and decreased on day 8 to day 19; thus, the concen-
tration of TNF-a in convalescent-phase samples from infants
with DHF/DSS was not significantly higher than that in samples
from control subjects (mean, 4.4 vs. 0.8 pg/mL; P p .1). Sig-
nificantly elevated IL-10 and IL-6 levels were detected in the
acute-phase samples from infants with DHF/DSS, compared
with samples from control subjects (mean, 73.8 vs. 0.3 pg/mL
[P p .000] and 28.2 vs. 1.4 pg/mL [P p .000], respectively).
In contrast, IL-10 and IL-6 levels in convalescent-phase samples
from infants with DHF/DSS decreased, compared with levels
in acute-phase samples, but were still significantly higher than
in samples from control subjects (mean, 8.3 vs. 0.3 pg/mL
[P p .002] and 22.7 vs. 1.4 pg/mL [P p .009], respectively).
However, further analysis showed that the mean serum levels
of IFN-g, TNF-a, IL-10, and IL-6 did not differ significantly
between patients with nonshock DHF and those with DSS (table

Dengue Hemorrhagic Fever in Infants JID 2004:189 (15 January) 227

 Table 5. Cytokine profile in acute-phase serum samples from 43 infants with nonshock dengue hemorrhagic fever (DHF),
19 infants with dengue shock syndrome (DSS), and 6 control infants.
Cytokine level, mean pg/mL  SD (range)
Infants with
Cytokine All infants nonshock DHF
IFN-g 56.2  115.4 (0690.8) 58.2  94.7 (0429.7)
TNF-a 9.0  13.2 (077.6) 9.0  14.0 (077.6)
IL-10 73.8  69.8 (2.8405.1) 72.0  52.7 (5.4206)
IL-6 28.2  41.7 (0210) 23.6  35.5 (2.1187.7)
IL-4 2.0  3.2 (017) 2.1  3.5 (017)
IL-2 2.6  4.7 (030) 3.2  5.3 (030)
NOTE. IL, interleukin; IFN, interferon; TNF, tumor necrosis factor.
a
All infants vs. healthy controls; calculated using the Kruskal-Wallis test.
b
Infants with nonshock DHF vs. infants with DSS; calculated using the Kruskal-Wallis test.
5) or between patients with primary and secondary infections
(data not shown). The mean serum levels of IFN-g, TNF-a,
and IL-10 in patients with fatal outcomes were not significantly
higher than those in survivors, but a significantly higher ele-
vation of IL-6 was observed in patients who died than in pa-
tients who survived the infection (mean, 131.2 vs. 23.0 pg/mL;
P p .007). On the other hand, compared with levels in samples
from control subjects, levels of IL-2 and IL-4 in acute- and
convalescent-phase samples in infants with DHF/DSS were not
elevated.
Coagulation tests and plasma protein C and S levels in
infants with DHF/DSS. Coagulation tests were done for 13
patients. Prolonged prothrombin time (PT) and activated par-
tial thromboplastin time (APTT), decreased fibrinogen levels,
and positive results of testing for d-dimer were seen in 5 pa-
tients (38.4%), 11 patients (84.6%), 10 patients (76.9%), and
2 patients (15.3%), respectively. PT and APTT were significantly
prolonged and fibrinogen levels were significantly lower in pa-
tients with DSS, compared with patients with nonshock DHF
(P p .008, P p .01, and P p .002, respectively; table 1).
Plasma levels of anticoagulant proteins C and S in the acute-
phase samples from infants with DHF/DSS were significantly
lower than levels in samples from healthy control subjects (mean,
54.5% vs. 105% [P p .000] and 55.4% vs. 110.9% [P p .000],
respectively). The levels of proteins C and S in the convalescent-
phase samples from infants with DHF/DSS were significantly
lower than levels in samples from control subjects (mean, 70.1%
vs. 105% [P p .000] and 82.5% vs. 110.9% [P p .000], re-
spectively). However, decreases in levels of proteins C and S
were not correlated with the severity of DHF (nonshock DHF
vs. DSS) or with GI bleeding (tables 1 and 2). Some correlation
between plasma levels of protein C (not of protein S) and serum
levels of AST and ALT (r p 0.34 [P p .02] and r p 0.37 [P
p .005], respectively) was indicated by linear regression analy-
sis. Linear regression analysis also showed significant nega-
tive correlations between protein C and S levels and PT (r p -
228 JID 2004:189 (15 January) Hung et al.
Infants with DSS Control infants Pa Pb
51.7  155.5 (0690.8) 4.1  5.8 (013.9) .01 .1
8.9  11.6 (037.2) 0.8  1.2 (02.1) .01 .9
77.8  100.2 (2.8405.1) 0.3  0.9 (02.3) .000 .4
38.6  52.8 (0210) 1.4  2.2 (04.9) .000 .5
1.6  2.6 (08.2) 0.2  0.5 (01.4) .2 .8
1.4  2.7 (010.4) 1.8  2.4 (06) .9 .1
0.7 [P p .004] and r p 0.8 [P p .02], respectively).
However, no association between levels of proteins C and S,
APTT, and fibrinogen levels was noted. Similarly, there was no
correlation between levels of proteins C and S and an increase
in Hct, which is a sign of capillary leakage (P p .2 and P p
.3, respectively).
Correlation of plasma concentrations of cytokines with se-
rum levels of transaminases and activation of coagulation.
In this study, serum IL-6 levels strongly correlated with PT
(r p 0.87; P p .001) but were not correlated with APTT or
fibrinogen levels (r p 0.53 [P p .1] and r p 0.46 [P p .1],
respectively) or with serum levels of transaminases. Further
analysis shows that serum IL-6 levels had some positive associ-
ation with the duration of fever in infants with DHF/DSS
(r p 0.31; P p .01). In contrast, serum IL-10 levels strongly
correlated with serum levels of AST and ALT (r p 0.91 [P p
.000] and r p 0.91 [P p .000], respectively) but were not re-
lated to PT, APTT, and fibrinogen levels. Moreover, serum TNF-
a levels were significantly associated with an increase in Hct
(r p 0.34; P p .005; table 6).
DISCUSSION
High fever (100% of patients), petechiae on the skin (99% of
patients), and hepatomegaly (97.1% of patients) were the most
common clinical findings associated with DHF/DSS in infants
in the present study. DSS (hypovolemic shock secondary to
capillary leakage) occurred in 22 (20.5%) of our 107 patients.
In contrast to children with DHF/DSS, in whom shock nearly
always accompanies or follows defervescence [16, 17], more
than one-half of the infants in the present study still had fever
at the time that shock occurred. As a result, physicians needed
to differentiate between DSS and septic shock. GI bleeding was
recorded in 8 infants (7.4%), 5 of whom received an FWB
transfusion. The finding that patients with GI bleeding had
significantly higher serum levels of AST and ALT than did those
Figure 2. Kinetic change of the levels of cytokines, by day after fever onset, in infants with dengue hemorrhagic fever (DHF)/dengue shock syndrome
(DSS). A, Levels of interferon (IFN)g in infants with DHF/DSS increased on day 4 to day 6 after fever onset and rapidly decreased on day 7 and
during the convalescent phase. B, Similarly, serum levels of tumor necrosis factor (TNF)a increased on day 4 to day 7 after fever onset in infants
with DHF/DSS and then decreased on day 8 to day 19. Interleukin (IL)10 (C) and IL-6 (D) levels in acute-phase samples were elevated; levels of
these cytokines in convalescent-phase samples decreased but were still significantly higher than those in controls. There was no significant elevation
in the levels of IL-2 (E) or IL-4 (F) in acute-phase and convalescent-phase samples, compared with controls.
 Table 6. Results of linear regression analysis showing sig-
nificant associations of cytokines with plasma levels of trans-
aminases and results of coagulation tests.
Cytokine, comparator b coefficiency r P
IL-6, coagulation test
PT 0.41 0.87
APTT 0.27 0.53
Fibrinogen 0.005 0.46
IL-10, transaminase
AST 17.8 0.91
ALT 3.07 0.91
TNF-a, increase in hematocrit 0.44 0.34
NOTE. ALT, alanine aminotransferase; APTT, activated partial throm-
boplastin time; AST, asparate aminotransferase; IL, interleukin; PT, pro-
thrombin time; TNF, tumor necrosis factor.
without GI bleeding suggests that hepatocellular damage may
underlie severe hemorrhaging. This has been observed in older
children and adults [18, 19]. Activation of the coagulation sys-
tem was indicated by prolonged PT (38.4% of patients) and
APTT (84.6% of patients), decreased fibrinogen levels (76.9%
of patients), and positive results of testing for d-dimer (15.3%
of patients). There was no significant difference in PT, APTT,
and fibrinogen levels in patients with and patients without GI
bleeding in the present study. As evidence that platelet abnor-
malities underlie severe hemorrhage, patients with GI bleeding
had significantly lower platelet counts than did those without
GI bleeding. Massive GI bleeding was often associated with a
grave prognosis; 3 of 4 infants with fatal outcomes had massive
GI bleeding.
Hemoconcentration, manifested by an increase in Hct of
20%, as defined by the WHO criteria [1], was observed in 98
patients (91.5%). Hct increased in the remaining 9 patients
(8.4%) by 10%19%; all but 1 of these patients had evidence
of capillary leakage, demonstrated by right-side or bilateral pleu-
ral effusion on chest radiography. Thrombocytopenia was ob-
served in 99 patients (92.5%). In a study of 31 Thai infants with
DHF, Witayathawornwong [8] reported that all patients had
thrombocytopenia and pleural effusion on chest radiography;
however, an increase in Hct of 120% was recorded in only 20
patients (64.5%), and another 11 patients (35.4%) had an in-
crease in Hct of 11%20%. The peak Hct, the increase in Hct,
and the lowest platelet count in infants with DHF/DSS in the
present study correlated with severity of the disease (nonshock
DHF vs. DSS; P p .007, P p .000, and P p .02, respectively).
Our data in the present study, as mentioned above, demonstrate
that clinical characteristics and laboratory findings for infants
with DHF/DSS are compatible with the WHO case definition,
which is based primarily on findings in older children.
Serologic responses, measured by IgM and IgG ELISAs, dem-
onstrated that almost all of our patients (95.3%) suffered from
230 JID 2004:189 (15 January) Hung et al.
primary dengue virus infections and that only 5 patients (4.7%)
had secondary infections. In our previous study, which included
47 infants with DHF/DSS, all patients had a primary infection
pattern detected by hemagglutination inhibition test [6]. Ninety-
eight (98.8%) of the 99 mothers of the infants in the study had
dengue virus antibodies, as shown by NS1 serotypespecific
.001
IgG ELISA. These results are consistent with the published
.1
observation that DHF/DSS in infants occurs during primary
.1
dengue virus infections [4, 5, 8, 9] and with the hypothesis that
severe disease is linked to transplacentally transmitted maternal
.000
dengue virus antibodies [4, 5]. In a large hospitalized series in
.000
Bangkok, Thailand, performed in 19621964, the vast majority
.005
of infants hospitalized with DHF had primary antibody re-
sponses measured in paired serum samples by the hemagglu-
tination inhibition test. A serologic survey of adults in Bangkok
showed that almost all women of child-bearing age had dengue
virus antibodies [4]. Kliks et al. [5] studied 13 infants from
whom DEN-2 viruses were isolated, all of whom had been
hospitalized for DHF and had well-documented primary den-
gue virus infections. All of the mothers of these infants were
immune to multiple dengue viruses; none had recently had
dengue virus infections. At birth, maternal dengue virus an-
tibody protects infants from dengue virus infection, but later,
the risk of development of DHF/DSS increases when IgG an-
tibodies are catabolized to lower titers. Continued catabolism
results in the loss of enhancing antibodies and a decrease in
risk for DHF/DSS at 1 year of age. This dual effect of passively
acquired antibodies also explains the age distribution of DHF/
DSS among infants in the present study. The largest numbers
of cases were in the 69-month age group. The age distribution
of DHF/DSS in infants in the present study is similar to that
in Thailand, Indonesia, and Myanmar [10].
Early diagnosis, proper treatment, and good nursing care are
the principles of management of DHF/DSS in infants, as well as
in children. Although the incidence of DHF/DSS in infants was
low (5%), the number of severe cases and the mortality rate
were high among infants, compared with among older children.
In the present study, almost all patients completely recovered,
but 4 patients died, resulting in a rather high case-fatality rate
of 3.7%. In comparison, the case-fatality rate was !1% among
all patients with DHF/DSS in the same time period [17, 20].
There are many reports that cytokine blood levels in patients
with DF differ from those in patients with DHF/DSS. All such
data are from studies of secondary dengue virus infections in
older children and adults [2128]. The present study is the first
to provide data on the serum levels of both proinflammatory
cytokines (IFN-g and TNF-a) and anti-inflammatory cytokines
(IL-10 and IL-6) in infants with DHF/DSS. Unusually high
levels of cytokines may play a role in the pathogenesis of DHF
in infants. To investigate whether there is any difference in the
cytokine profiles of infants with primary dengue virus infections
and older children with secondary infections, we measured the
concentrations of cytokines in 39 children with DHF/DSS aged
415 years who had secondary dengue virus infections, using
the same techniques described in the present study. We found
significant elevations of serum IFN-g and IL-10 levels in chil-
dren with DHF/DSS (mean  SD, 109.5  225.7 and 78.9 
57.6 pg/mL, respectively), compared with levels in control sub-
jects (mean  SD, 6.2  6.2 and 0.5  1.1 pg/mL, respectively).
Serum levels of TNF-a, IL-6, IL-4, and IL-2 were not elevated
in children with DHF/DSS who had secondary dengue virus
infections (mean  SD, 2.5  2.1, 8.8  6.7, 1.0  1.2, and
0.3  0.9 pg/mL, respectively), compared with levels in control
subjects (mean  SD, 1.2  1.4, 3.3  2.3, 0.3  0.7, and 1.3
 1.6 pg/mL, respectively; unpublished data). The levels of IFN-
g and IL-10 in children with secondary dengue virus infections
did not differ from those in infants with primary dengue virus
infections. Instead, the cytokine profile differed. In contrast to
children with DHF/DSS, who produce elevated levels of IFN-
g and IL-10, infants produced elevated levels of TNF-a and
IL-6, in addition to IFN-g and IL-10. It is evident that, in
opposition to the view of Rothman and Ennis [12], cross-
reactive T cells that are activated in response to secondary
infection with a different serotype are not required to produce
the high levels of cytokines that accompany severe DHF. A
unifying hypothesis is that enhancing antibodies acquired pas-
sively from mothers (primary infection in infants) or actively
from previous infection (secondary infection in children) pro-
motes dengue virus infection of Fc-bearing cells, resulting in a
large, infected cell mass. T cell and cytokine responses are sim-
ply proportional to the infected cell mass. Levels of the inflam-
matory cytokine IFN-g and the anti-inflammatory cytokine IL-
10 are elevated simultaneously in patients with DHF/DSS.
Infants experiencing DHF during primary dengue virus infec-
tion also have elevated blood levels of TNF-a and IL-6.
Linear regression analysis revealed that levels of cytokines
such as TNF-a, IL-6, and IL-10 are correlated with some bio-
logical responses. An elevated serum level of TNF-a has a sig-
nificant correlation with the increase in Hct that is a sign of
capillary leakage in infants with DHF/DSS. This may be the
result of TNF-a toxicity to vascular endothelial cells and may
increase vascular permeability. There were significantly higher
levels of IL-6 in infants with fatal outcome, compared with
levels in those who survived the infection (P p .007 ). But no
significant difference exists between patients with and patients
without shock. The serum level of IL-6 in infants with DHF/
DSS was also strongly correlated with activation of the extrinsic
pathway of coagulation (PT; P p .001) but not with the in-
trinsic pathway (APTT) or with fibrinogen levels. This is con-
sistent with reports that IL-6 levels are positively associated
with the severity of dengue virus infection in both children and
adults [29]; of increased serum levels of IL-6 and IL-8 in pa-
Dengue Hemorrhagic Fever in Infants JID 2004:189 (15 January) 231
tients with DHF, but not in patients with DF [30]; that the
highest levels of IL-6 are found in patients with DSS [31]; and
that IL-6 is significantly associated with the activation markers
of coagulation and fibrinolysis [32]. Furthermore, a significant
and strong correlation between IL-10 and serum levels of AST
and ALT was found in infants with DHF/DSS. Green et al. [33]
also reported that elevated levels of IL-10 in children with den-
gue virus infection were associated with disease severity (DF
vs. DHF) and the degree of capillary leakage, quantified by the
size of pleural effusions. The elevation of IL-6 and IL-10 levels
in infants with DHF/DSS suggests that, when an infant responds
to dengue virus infection via generation of inflammatory cyto-
kines (IFN-g and TNF-a), there is simultaneous generation of
inhibitory cytokines (IL-6 and IL-10) to counteract the inflam-
mation. The more stimulation of the pathogen, the higher the
levels of stress mediators, such as IL-6 or IL-10, induced in the
host to counteract the response. Cytokines can cause cell acti-
vation synergistically or antagonistically; the net outcome will
depend on the balance between various cytokine actions [13].
The present study emphasizes that DHF/DSS in infants is
not uncommon and that clinical characteristics and laboratory
findings for infants with DHF/DSS are compatible with the
WHO case definition for DHF/DSS in children. This study is
the first to describe cytokine blood levels during the acute stage
of DHF/DSS in infants. The cytokine profile in infants with
primary dengue virus infections and children with secondary
infections were studied in the same laboratory, using the same
assay methods. Overproduction of both proinflammatory cy-
tokines (IFN-g and TNF-a) and anti-inflammatory cytokines
(IL-10 and IL-6) may play a role in the pathogenesis of DHF/
DSS in infants.
Acknowledgments
We thank Tran Tan Tram, the Director of the Childrens
Hospital No. 1Ho Chi Minh City (Ho Chi Minh City, Viet-
nam), and Chung-Ming Chang, National Health Research In-
stitutes (Taiwan), for help and support during the course of
this study. Thanks also to Hoang Le Phuc, for help with data
analyses, and the doctors and nurses of the Department of
Dengue Hemorrhagic Fever, Childrens Hospital No. 1Ho Chi
Minh City, for providing excellent patient care.
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