322

Dengue in the Early Febrile Phase: Viremia and Antibody Responses

David W. Vaughn, Sharone Green, Siripen Kalayanarooj,
Bruce L. Innis, Suchitra Nimmannitya,
Saroj Suntayakorn, Alan L. Rothman, Francis A. Ennis,
and Ananda Nisalak

Department of Virology, Armed Forces Research Institute of Medical

Sciences, Bangkok, Bangkok Childrens Hospital, Bangkok, and
Kamphaeng Phet Provincial Hospital, Kamphaeng Phet, Thailand;
Division of Infectious Diseases and Immunology, University of
Massachusetts Medical Center, Worcester, Massachusetts; Department
of Virus Diseases, Walter Reed Army Institute of Research,
Washington, DC

Dengue is a major cause of morbidity in tropical and subtropical areas. It is estimated that 250,000 cases of severe dengue
infections, called dengue hemorrhagic fever (DHF), and at least
100 million cases of uncomplicated dengue fever (DF) occur
annually [1]. Dengue viruses consist of four serotypes and are
transmitted by mosquitoes, particularly Aedes aegypti [2]. DF
in adults is typically a flu-like illness characterized by fever,
headache, eye pain, myalgia, arthralgia, and rash [3, 4]. Most
dengue infections in children are mild or asymptomatic but
may present as undifferentiated fever, classic DF, or DHF [5,
6]. DHF includes plasma leakage that may lead to shock, and
defects in hemostasis including thrombocytopenia that may
result in hemorrhage. Mortality rates for hospitalized DHF patients in dengue-endemic areas range from 1% to 40% [7].
There is no treatment other than careful fluid management
[8]. No preventive measures are available beyond mosquito

Received 2 October 1996; revised 21 January 1997.

Presented in part: 44th Annual Meeting of the American Society of Tropical
Medicine and Hygiene, San Antonio, Texas, November 1995 (abstract 159).
Written informed consent was obtained from parents or guardians of patients
who met enrollment criteria. The study was approved by the Ethical Review
Subcommittee of the Ministry of Public Health, Thailand, the Institutional
Review Board of the University of Massachusetts Medical Center, and the
Human Subjects Research Review Board of the Office of the US Army Surgeon
General, Washington, DC.
The opinions or assertions contained herein are the private ones of the
authors and are not to be construed as official or as reflecting the views of the
US government or the Ministry of Public Health, Thailand.
Financial support: US Army Medical Research and Materiel Command and
NIH (AI-34533).
Reprints or correspondence (from the USA): David W. Vaughn, USAMCAFRIMS, APO AP 96546.
The Journal of Infectious Diseases 1997;176:32230
q 1997 by The University of Chicago. All rights reserved.
00221899/97/76020003$02.00

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avoidance. Dengue is an emerging disease throughout tropical

and subtropical regions as the territory of its principal vector,
A. aegypti, expands to include Asia, Africa, Central America,
South America, and the Pacific [9].
Preexisting dengue cross-reactive antibodies and T cells are
central to the immune enhancement theory of dengue pathogenesis, which attempts to explain why sequential or secondary
dengue infections are more likely to result in severe disease
than first or primary infections. According to this hypothesis,
cross-reactive nonneutralizing antibodies from a previous heterologous dengue infection facilitate virus entry into Fc receptor bearing cells such as monocyte/macrophages. This increase in the number of antigen-presenting cells activates
precursor cross-reactive memory T cells, which are present in
high numbers as a result of previous heterologous infection.
This leads to the release of chemical mediators that cause
plasma leakage and results in increased disease severity
[10, 11].
The purpose of this study was to characterize the early virologic and antibody responses of dengue infection in an effort
to better define the pathophysiology of dengue disease. This
clinical investigation examined several aspects of dengue
pathogenesis: Are the duration and level of viremia related to
outcome? Do certain dengue virus serotypes cause more severe
disease than others? Do the kinetics of the antibody response
early in infection correlate with the severity of disease? Detailed clinical and cellular immunologic aspects of this study
are reported elsewhere [12] (Green S, et al., unpublished data).
Patients and Methods
Study design. Children with fever for 3 days without localizing signs of infection were enrolled and were observed in

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A multicenter effort was begun in 1994 to characterize the pathophysiology of dengue using a
study design that minimized patient selection bias by offering enrollment to all children with
undifferentiated fever for 72 h. In the first year, 189 children were enrolled (age range, 8 months
to 14 years). Thirty-two percent of these children had dengue infections (60 volunteers). The percentage of children with a secondary dengue infection was 93%, with only 4 (7%) having a primary
dengue infection. The virus isolation rate from the plasma of children with dengue was 98%. Viremia
correlated highly with temperature. All four dengue virus serotypes were isolated at both study
sites. This study demonstrates that all four serotypes of dengue virus can cause dengue hemorrhagic
fever, that all dengue patients as defined by serology experience viremia during the febrile phase,
and that as fever subsides, so does viremia.

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Viremia and Antibody Responses in Dengue

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all patients during the first 3 study days and then with the remaining
specimens from patients whose plasma samples contained virus
during the first 3 days. In brief, viremia was assessed by injecting
20 live mosquitoes with 0.34 mL of undiluted patient sera. After
14 days, 15 of the surviving mosquitoes were tested for flavivirus
antigen by IFA of the head [16]. The number of positive tests
over the number of mosquitoes tested was recorded. Virus-positive
mosquitoes were used to infect C6/36 cell cultures for identification of virus type using a panel of monoclonal antibodies against
dengue and Japanese encephalitis virus (JEV) in an EIA [17].
Antibody responses. IgM and IgG to dengue and JEV were
measured in all specimens by antibody capture EIA [18]. For single
specimens, 40 U of IgM to dengue (with dengue IgM greater than
JEV IgM) was considered evidence of a dengue infection (30 U
if from paired sera with 15 U of antibody in the acute specimen).
A dengue IgM-to-IgG ratio 1.8:1 defined a primary dengue infection. A ratio 1.8:1 defined a secondary dengue infection. With
serial specimens, a 2-fold increase in IgG to dengue with an absolute value of 100 U indicated a secondary infection in the absence
of IgM to dengue of 40 U.
Hemagglutination-inhibition (HAI) antibody against dengue virus types 14 and JEV were measured in all specimens [19]. A
4-fold increase was considered positive for acute flavivirus infection. The infection was diagnosed as primary if titers 1 week
after onset of illness were 1:1280 or as secondary if antibody
titers were 1:1280 [13].
A serologic diagnosis was assigned to each patient on the basis
of EIA and HAI results. Patients in whom the serologic diagnosis
was suggestive of a recent (rather than acute) dengue infection
(falling IgM level, fixed HAI titer of 2560, or both) or for
whom no convalescent specimen was available and no virus was
isolated were diagnosed as unknown and were excluded from
the analysis.
Statistical analysis. Data were entered and manipulated using FoxPro for Windows software (Microsoft, Redmond, WA)
and analyzed using SPSS for Windows version 6.1.3 (SPSS,
Chicago, IL).

Results
Children enrolled. The total enrollment at Bangkok Childrens Hospital and Kamphaeng Phet Provincial Hospital between 24 April and 14 December 1994 was 189 volunteers.
The distribution by clinical diagnosis and corresponding demographic information are shown in table 1. Sixty of the 189
children had acute dengue infection.
Virus isolation. A dengue virus was isolated from the
plasma of 59 of the 60 dengue patients (table 2). The remaining
patient (KPP94-029) had clear serologic evidence for an acute
secondary dengue infection; he defervesced within 3 h of venipuncture for the first blood sample, which had elevated antibody levels (dengue IgM 95 U, HAI 1:1280). Of the 129
children who did not have serologic test results diagnostic of
acute dengue infection, none had a dengue virus isolated.
Virus identification. The serotypes of the 59 dengue viruses
isolated are shown in table 3. All 4 serotypes of dengue virus

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hospital until 1 day following defervescence. Data were collected

and treatment was provided according to standard guidelines.
Blood samples were obtained daily up to a maximum of 5 consecutive collections, and a follow-up specimen was obtained 8
days after enrollment. A right lateral decubitus chest radiograph
was obtained on the day after defervescence. Children who had
antibody responses diagnostic of an acute dengue virus infection
had additional blood samples collected at 6 months and 1 year
after enrollment.
Definitions. Study day 1 is the day on which a child was
enrolled in the study and the first blood sample was obtained.
Fever day 0 is the day of defervescence, that is, the day that the
temperature fell below 387C without further significant temperature
elevation. Days prior to fever day 0 are designated fever day 01
(1 day before defervescence), fever day 02, etc. The day after
defervescence is fever day 1. Assessment of disease severity followed the World Health Organization grading system [13]. Children with a confirmed diagnosis of dengue based on viremia or
antibody responses (or both) without evidence of plasma leakage
were considered to have DF; those with a fall in platelets and
plasma leakage manifested by either a 20% increase in hematocrit,
a pleural effusion, or ascites without shock were diagnosed as
having DHF grade 1 (DHF-1, no spontaneous hemorrhage) or
DHF-2 (spontaneous hemorrhage). Dengue patients experiencing
peripheral vascular collapse with a pulse pressure of 20 mm Hg
or clinical signs of shock (or both) were considered to have DHF3; those with undetectable blood pressure were diagnosed as having DHF-4. Other febrile illnesses (OFI) were defined as cases
with no antibody evidence of dengue infection, no isolation of
dengue virus, and no obvious bacterial, rickettsial, or protozoal
etiology. OFI was a diagnosis of exclusion; most illnesses were
self-limited and presumed to be viral infections other than dengue.
Selection of patients. Pediatric patients seen at the Bangkok
Childrens Hospital (a 538-bed tertiary-care facility) and the Kamphaeng Phet Provincial Hospital (a 335-bed secondary-level facility 270 km north of Bangkok) from 24 April to 14 December 1994
were enrolled. Dengue is transmitted year-round in Thailand but
with a distinct seasonal peak each August through October. Children enrolled were in stable condition and were early in the course
of a nonspecific febrile illness [12]. Briefly, febrile children were
evaluated consecutively to identify those who met screening criteria (age 6 months through 14 years, weight 6 kg, temperature
38.57C, history of fever for 72 h, and flushed face). Patients
meeting screening criteria were examined by a study physician to
exclude those with fever 72 h, focal source of infection (e.g.,
otitis media, pneumonia, meningitis), coryza, chronic illness including anemia, or unstable vital signs. At each hospital, a maximum of 6 children were enrolled per week.
Data and specimen collection. Study participants were examined daily by a study physician, and a case report form was completed. Daily blood specimens were collected into EDTA anticoagulant and maintained on wet ice until received in the processing
laboratory. There, while at 47C throughout, samples were centrifuged at 300 g for 10 min, the resulting platelet-rich plasma was
recentrifuged at 800 g, and the platelet-poor plasma supernatant
was frozen at 0707C until assays were performed.
Virus isolation. Virus isolation in Toxorhynchites splendens
mosquitoes [14, 15] was attempted with each plasma sample from

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Vaughn et al.

Table 1. Diagnosis and demographic information for enrolled patients.

Diagnosis

No.

Dengue fever
Dengue hemorrhagic fever
Grade 1
Grade 2
Grade 3
Bacterial*
Other febrile illness
Unknown
Totals

Mean age, years

(range)

Sex
(% male)

32

8.1 (2 14)

47

5
14
9

6.0 (1 9)
8.7 (2 11)
8.2 (4 11)

60
71
44

7.0
6.2
5.7
6.8

33
55
43
53

3
112
14
189

(1 14)
(0.7 14)
(0.8 11)
(0.7 14)

were isolated from patients at each hospital. There was no

temporal pattern of virus type isolated at either hospital. At
Bangkok Childrens Hospital, dengue virus types 1, 2, and 4
were isolated in at least 5 of the 8 study months and type 3
was isolated from August through November. In Kamphaeng
Phet, types 2 and 4 were isolated from April through July.
Dengue virus type 3 was isolated in May and June, and the
single type 1 virus was isolated in September. There were no
dengue isolates in Kamphaeng Phet after September in this
study.
All four dengue virus types caused both DF and DHF. No
correlation between virus type and disease severity could be
demonstrated (table 3). While types 2 and 3 were almost twice
as likely to cause DHF rather than DF in this sample, the
difference was not significantly different (Pearsons x2, P
.3). In comparing the amount of plasma leakage, judged by the
level of the pleural effusion in a right lateral decubitus chest
radiograph 1 day after defervescence, there was no difference
among dengue virus serotypes (analysis of variance, P .3).
Kinetics of viremia. The blood samples of children who
had serologic evidence of an acute dengue infection were dengue virus positive in nearly all inoculated mosquitoes early in
the illness. As an estimate of the virus titer, the mean percentage
of inoculated T. splendens mosquitoes that were positive for
virus was calculated. The percentage of inoculated mosquitoes
that were positive decreased as fever day 0 approached (figure
1). Viremia correlated highly with temperature (Spearmans
correlation coefficient, P .001). All blood specimens collected 2 days prior to defervescence yielded a virus, whereas
all specimens collected 2 days following defervescence did
not. There was an abrupt and rapid disappearance of virus by
live mosquito culture at the time of defervescence.
Duration of viremia. The duration of viremia was calculated by assuming that a patient was continuously viremic from

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the reported onset of fever through the day of the last positive
culture. For 7 patients, the last day of viremia, when no inoculated mosquitoes were IFA-positive, was not identified. In 44%
of the remaining patients, the percentage of mosquitoes positive
was 100% on the day prior to the day that no virus was
isolated. In 56% of the patients, the percentage of mosquitoes
positive fell from 100% to 0% in 24 h. Therefore, it was
assumed for the 7 patients for whom the percentage of mosquitoes positive was 100% on the last day measured that the
viremia would persist for another half day. The mean number
of days of viremia for primary cases was 5.4 days (95% confidence interval [CI], 4.0 6.7) ending on fever day 1.6 (range,
0 2.5), and the mean number of days of viremia for secondary
cases was 4.5 days (95% CI, 4.3 4.8) ending on fever day 0.6
(range, -2 to 2.5). Mean duration of fever for patients with
primary infections was 3.8 days (95% CI, 1.4 6.1), and for
patients with secondary infections the mean was 4.1 days (95%
CI, 3.8 4.4). The duration of viremia did not correlate with
dengue virus serotype or disease severity (grade).
Antibody responses. Fifty-nine of the 60 dengue patients
were diagnosed with dengue infection by antibody responses
(table 2). One remaining patient (KPP94-011) had a dengue
virus type 4 isolated in the single blood specimen collected 2
days before defervescence; no convalescent blood sample was
available for antibody analysis. Only 4 of the 59 children with
diagnostic antibody responses had primary infections (e.g.,
figure 2A), and the rest had secondary infections (e.g., figure
2B). All 4 of the patients with primary dengue infections had
DF, but 49% of patients with secondary dengue infections had
DHF.
There was excellent agreement between EIA and HAI for
diagnosing acute dengue infection. Of the 55 secondary cases,
the rise in IgM to dengue did not meet test criteria for an acute
dengue infection in 3 (change from 15 to 28 U; 0 to 17 U; and
5 to 22 U). However, in each of these 3 patients, the rise in
IgG to dengue was dramatic (change from 41 to 143 U; 33 to
310 U; and 10 to 289 U, respectively). HAI titers in all 3
patients rose to 1:5120, and dengue virus was isolated from
the blood. Thus, all secondary dengue infections had positive
serologic test results by both EIA and HAI. The time sequence
for serial specimens to be considered positive for acute secondary dengue infection is shown in figure 3. All patients with
secondary dengue infections acquired diagnostic levels of anti-

Table 2. Dengue virus isolation rate by serologic diagnosis.

Serology
Primary infection
Secondary infection
Nondiagnostic
Total

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No. of
virus isolates

Isolation
rate (%)

4/4
54/55
1/1
59

100
98
100
98

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* Haemophilus influenzae meningitis, Salmonella gastroenteritis, urinary

tract infection.

Nondengue self-limited febrile illness; presumed viral.

Inadequate specimens to rule out dengue (n 10) or recent dengue infection rather than acute (n 4).

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Viremia and Antibody Responses in Dengue

325

Table 3. Serotypes of dengue viruses isolated from patients at the two study hospitals, by grade of
illness.
Bangkok
Childrens
Dengue virus
serotype
1
2
3
4
Totals

Kamphaeng Phet

Both hospitals

DHF

DF

DHF

DF

Total viruses

% DHF

3
5
5
5
18

8
3
2
5
18

1
5
1
2
9

0
4
2
8
14

12
17
10
20
59

33
59
60
35
46

NOTE. DHF, dengue hemorrhagic fever; DF, dengue fever.

The relative reactivity of IgM for the dengue virus complex

was evaluated by determination of levels of IgM to equivalent
amounts of tetravalent dengue antigen and JEV antigen. JEV
is the only other flavivirus known to infect people in Thailand. EIA identified each of the 60 flavivirus infections as
dengue rather than JEV. With 1 exception, the ratio of dengue
IgM to JEV IgM in the follow-up specimen (n 49) was 1,
ranging from 1.3 to 22 (mean, 4.8), supporting a serologic
diagnosis of dengue rather than Japanese encephalitis (JE). This
ratio for the remaining patient was 0.93 on fever day 4, although
it had been 1.3 on the day of defervescence (fever day 0) and
even higher earlier in the illness (4.3, 2.6, and 2.9 on fever
days 03, 02, and 01, respectively). The ratio of dengue IgM
to JEV IgM was most useful to distinguish dengue from JE
from fever day 0 onward (figure 4). Levels of IgG against

Figure 1. Mean values by fever day for 55 patients with secondary dengue infections for maximum daily temperature, % of mosquitoes
positive by indirect IFA for dengue antigen, dengue IgM and IgG antibody levels, and reciprocal titer of hemagglutination-inhibition antibody
(HAI) (average of all 4 dengue types, log scale).

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body by EIA by 1 day following defervescence in serial testing.

If assay results were considered individually without the benefit of previous results (single specimen analysis), the EIA was
diagnostic in just 29% (15/51) of patients on fever day 0 and
67% (28/42) on fever day 1 (in a single specimen, IgM antibody against dengue must be 40 U). Diagnostic serology
by HAI (79% positive on fever day 1) lagged behind that by
EIA. This is because a diagnosis of secondary dengue infection based on HAI antibody responses requires a rising titer
to 1:1280. None of 4 cases of primary dengue infections
acquired diagnostic levels of dengue antibody by fever day
1. IgM antibody, as measured by EIA, developed later in
primary infections, and for a diagnosis by HAI, a specimen
must be obtained at least 1 week into the illness with a maximum titer of 1:1280.

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Vaughn et al.

JID 1997;176 (August)

illness). However, IgM antibody was elevated on fever day 6

and HAI antibody was present in the follow-up specimens at
6 and 12 months, which had highest antibody titers against the
infecting serotype of virus.
The timing of the antibody increases early in secondary
dengue infections is shown in figure 1. The HAI antibody titer
increased before the antibodies measured by EIA. The IgG

Figure 2. Typical early viremia and antibody responses: A, primary

dengue (study no. CHD94-090, dengue virus type 1 [DEN-1] was
isolated); B, secondary dengue (study no. KPP94-007, DEN-2 virus
was isolated); and C, recent secondary dengue infection (CHD94101, no virus isolated). First fever day shown is first day of illness.
Fever day 0 is day of defervescence. / days on which virus was
isolated. HAI hemagglutination-inhibition antibody assay.

dengue and JEV were similar (r .94); therefore, they failed

to distinguish between infection with dengue viruses and JEV.
The HAI antibody responses to homologous and heterologous dengue virus antigens were similar. The HAI antibody
responses to JEV antigen commonly lagged behind the response to dengue antigens (figure 2B). Among the four primary
infections, the homologous antibody response was the first to
appear in 2 patients and first along with antibody to two other
serotypes in 1 patient (figure 2A). In the fourth patient, HAI
antibody was not seen on fever day 6 (9 days after onset of

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Figure 4. Mean ratio of IgM to dengue (EIA units) to IgM to

Japanese encephalitis virus (JEV) with 95% confidence intervals by
fever day (fever day 0 day of defervescence). Reference (dashed)
line is at 1.0, where levels of IgM to dengue and to JEV are about
equal.

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Figure 3. % of 55 patients with acute secondary dengue infections

acquiring diagnostic levels of dengue antibody by fever day and assay.
Plasma was collected daily through fever day 1 and again on study
day 8 per study protocol (here grouped as fever day 6). All 4 children
with primary dengue infections acquired dengue antibody levels consistent with acute dengue infection after fever day 1 and are not
included in this figure. Fever day 0 day that temperature dropped
to 387C. EIA measured IgM and IgG antibody against dengue viruses. HAI hemagglutination-inhibition antibody assay against 4
serotypes of dengue virus.

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Viremia and Antibody Responses in Dengue

Discussion
In this study of children with acute fever, all children who
had a dengue virus isolated from their blood had antibody
responses consistent with an acute dengue virus infection, and
all children with an acute dengue antibody response had detect-

Figure 5. Mean values of hemagglutination-inhibition antibody

(HAI) reciprocal titer and IgG and IgM to dengue on fever days 03
(n 21), 6 (n 45), 180 (n 48), and 365 (n 48); secondary
infections only.

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able viremia (with the exception of 1 child who was evaluated

3 h prior to defervescence). Our data demonstrate the occurrence of viremia early in the febrile period of dengue and its
rapid elimination as fever abates and antibody levels rise.
Ashburn and Craig [20] working in Manila in 1906 first
demonstrated that patients with dengue infections were viremic
early in their illness. Subsequently, virus isolation was confirmed to be more likely early in the illness [21]. Kuberski et
al. [22] isolated dengue virus type 1 in 73% of patients on the
first day of illness, with decreasing rates until no isolations
were made after 6 days of illness. Higher isolation rates, from
51% to 100%, have also been reported for patients experiencing
first or primary dengue infections [23, 24]. After the first day
of illness, Kuberski et al. [22] was more likely to isolate virus
and demonstrate higher virus titers in children experiencing
primary dengue infections. While Gubler et al. [24] also noted
that virus isolation was more common in primary dengue infections, they found that among dengue type 3 infections, the titer
of type 3 virus was 5-fold higher in secondary infections.
Our data confirm and extend previous data on the relative
ease of virus isolation early in the illness. Our study has
several features that probably explain the high rate of viremia
we found compared with most published studies of DF and
DHF [23 27]. We followed children with fever and obtained
serial blood samples from the same patient during the course
of illness. Furthermore, children were enrolled in the study
shortly after the onset of illness, during the febrile phase when
virus isolation is more likely. On the other hand, Gubler et
al. [28] isolated dengue type 2 virus from only 6 of 18 patients
on the island of Tongatapu in 1974 despite specimen collection early in the illness. In that study in the Kingdom of Tonga
and another study by the same group in central Java in 1978,
specimens were collected (live Aedes aegypti mosquitoes fed
on patients) during the first days of illness over several days,
yet the isolation rate was only 33% [28, 29]. The illnesses in
both the 1974 and 1978 outbreaks were remarkable for their
mildness and relative short duration; the lower isolation rate
may represent either a difference in virus isolation technique
or the presence of a less virulent strain of dengue type 2 virus,
resulting in lower virus titer and mild disease. Partial genome
sequencing of the 1974 Tonga dengue type 2 virus has shown
it to be significantly different from Thai isolates including
isolates from our study [30, 31] (Rico-Hesse R, personal communication).
The typical duration of viremia in our patients was 4 5
days, as has been seen previously [32]. While not statistically
significant, our data are consistent with the findings of Kuberski
et al. [22] and Gubler et al. [32] in showing that viremia persists
longer in children experiencing primary infections rather than
secondary dengue infections. Our results suggest that this may
be due to continued viremia into the afebrile phase, because
viremia lasted 1 day longer than the duration of fever in primary
dengue patients. If viremia extends further into the afebrile

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antibodies as measured by EIA increased before IgM and rose

to higher peak levels.
Virus-antibody interactions. As antibodies increased, the
virus titer, as estimated by the percentage of mosquitoes positive for dengue virus, decreased (figure 1). The first day in
which virus isolation was not possible was identified in 46
patients with secondary dengue infections. For the remainder,
a dengue virus was isolated on the last day that blood was
obtained during the acute illness. On the last day a virus was
isolated, the mean level of IgM antibody against dengue virus
was 19 U (range, 0 123) and the geometric mean HAI antibody
titer (average of all four dengue types) was 1:140 (range,
1:10 1:2560). On the first day a virus was not isolated, the
mean level of IgM against dengue virus was 46 U (range, 2
160), and the geometric mean HAI titer was 1:860 (range,
1:24 1:7240).
Long-term antibody responses. The long-term IgG, IgM,
and HAI titers for the 55 secondary dengue patients are shown
in figure 5, with data at fever days 03, 6 (during the second
week of illness), 180, and 365. Dengue IgM antibodies fell to
low levels by 6 months (mean, 9 U; range, 0 40). All children
with secondary dengue infections had intermediate HAI antibody levels at 6 months (mean titer, 1:174; n 48) and at 12
months (mean titer, 1:82; n 48). The 4 children with primary
dengue infections had lower levels of HAI antibody at 6 months
(mean titer, 1:17) and 12 months (mean titer, 1:18).

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ting serologic results from single specimens or failing to perform virus isolation (or both). However, in a nonresearch
setting, patients often present later in the course of their illness, and a single high-titered specimen can be assigned a
statistical probability of being associated with an acute secondary dengue infection. In addition, the consistent timing of
fever, viremia, and antibody responses seen in the present
study as well as the other clinical indicators of dengue illness
[12] document that the children experienced acute illnesses
due to dengue virus infection rather than illness due to another
etiology with incidental dengue virus infection.
Concerning laboratory diagnosis of acute dengue virus infection, the message of this study is that if the patient with dengue
has fever, then attempts at virus isolation (or dengue antigen
detection or genome identification) should be successful while
serologic assays may still be negative; conversely, if fever has
abated, then virus isolation is less likely to be successful and
serology is more likely to be diagnostic.
Previous studies of dengue HAI antibody duration following
natural infection have been performed as cross-sectional antibody surveys in areas where clinical dengue has been absent
for some time. These studies suggest that HAI antibody against
dengue virus can be long-lived: Theiler et al. [39] and Rosen
[40] found HAI antibody against dengue virus 30 and 45 years,
respectively, after the dengue outbreaks of 1927 and 1928 in
Greece; Rosen [41] also found HAI antibody 12 years after an
epidemic in Panama; and Hammon et al. [42] found HAI antibody against dengue type 2 24 years after a dengue epidemic
in 1934 in Florida. The previous studies assumed the HAI
antibodies were due to dengue virus infection because the epidemic was clearly dengue on the basis of clinical criteria, and
persons born since the epidemic did not have antibody. HAI
antibody has also been shown to be long-lived in persons who
were experimentally infected with dengue viruses by allowing
infected mosquitoes to feed on them. Halstead [43] demonstrated HAI antibody 42 48 years following experimental dengue infections in volunteers infected in the Philippines between
1924 [44] and 1930 [34]. The present study followed HAI
antibody levels in a prospective fashion to 1 year after infection
and showed a slow decline in antibody titer between 6 and 12
months. We plan to follow children in this study yearly for at
least 3 years after infection to further define long-term HAI
antibody responses to dengue viruses.
All 4 children experiencing primary dengue infections were
classified as having DF compared with secondary patients, of
whom 50% demonstrated clear evidence of plasma leakage.
These results are consistent with prior epidemiologic studies
of severe dengue illness in secondary cases. In this prospective
study, we demonstrated viremia in 98% of patients early in the
course of disease. Further studies will be needed to validate
major tenets of the immune enhancement theory, that is, the
effects of preexisting cross-reactive antibody and T cell responses at the time of the secondary infection and the impor-

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period in primary dengue infections, it may be due to more

gradual antibody or cellular immune responses.
Some observers have found that certain serotypes of dengue
virus (serotypes 2 and 3) are more likely to cause severe
disease [23, 29, 33]. However, Gubler et al. [24] could not
demonstrate a relationship between dengue serotype and disease severity in Indonesia. In these and other earlier studies,
a possible confounder arises from the generally low isolation
rates of 15% 65% [23 27]. If, for example, dengue type 2
virus is easier to recover than the other serotypes of dengue
virus, then more severe disease would be falsely biased toward
dengue type 2.
In the present study, all four dengue virus serotypes were
isolated during a single season in an urban childrens hospital
and a rural provincial general hospital. The circulation of three
or more serotypes of dengue throughout Thailand has been
observed consistently for 2 decades. This promotes the occurrence of sequential dengue infections and appears to sustain
endemic DHF. The isolation of virus from nearly 100% of
patients removes one potential bias in evaluating differences in
disease outcomes by infecting dengue serotype. While patients
infected with serotypes 2 and 3 were nearly twice as likely to
progress from DF (undifferentiated febrile illness) to DHF in
our study, this difference was not statistically significant. Continued enrollment in this study may allow adequate numbers
of patients to provide a more definitive answer.
Studies of antibody responses in dengue began in 1930 with
the development of a complement fixation test [34]. The use
of HAI antibody assays against all four dengue virus serotypes
in the early 1960s demonstrated that HAI antibody responses
were often cross-reacting among the four. The results of HAI
assays were used to distinguish between primary and secondary
dengue infections [35 37]. More recent studies of antibody
kinetics show that, in primary infections, IgM to dengue viruses
increases to high levels in nearly all patients within 2 days
of defervescence and peaks within 2 weeks. In nonprimary
(secondary, tertiary, etc.) infections, the IgM response is variable, sometimes absent or lagging behind a dramatic increase
in IgG antibodies [18, 38].
In the present study, both EIA and HAI antibody results
were efficient in the diagnosis of acute dengue virus infections
and were able to identify the infection as primary or secondary. In Southeast Asia, EIA had the further advantage of being
able to distinguish dengue from JEV infections. Four patients
(2%) not included in the analysis had serologic evidence of
a recent rather than an acute dengue infection, that is, a fixed
HAI titer of 2560 [13] or EIA antibody levels that were
high and level or falling within 72 h of the onset of illness
(e.g., figure 2C). Given that 90% of dengue infections in Thai
children are mild or asymptomatic [6], these children probably
experienced an asymptomatic dengue infection or mild dengue illness prior to the febrile illness that brought them to the
hospital. These patients illustrate the difficulties in interpre-

JID 1997;176 (August)

JID 1997;176 (August)

Viremia and Antibody Responses in Dengue

tance of the virus burden and immunologic responses in secondary infections that lead to DHF.

Acknowledgments

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We thank Suntaree Ratanachu-eke, Wanida Suteewarn, Narongchai Kunentrasai, Wiboon Viramitrchai, Supaporn Kiatpolpoj, and
the pediatric nurses of Bangkok Childrens Hospital and Kamphaeng Phet Provincial Hospital for supervising data collection
and providing exceptional patient care; Nathada Plavooth, Pranom
Vangnai, Somnuk Lumjiak, Sumetha Hengprasert, Sumolvadee
Saravasee, and Wipa Chawachalasai for specimen and data collection; Somkiat Changnak, Choompun Manomuth, Aree Na-Nongkai, Pranee Saisang, Somsamai Tapana, Songdej Saengsi, and
Weerasak Yiphu for specimen processing; Nonglak Ongsakorn
and Sanguan Boonak for virus isolation; Naowayubol Nutkumhaeng and Somsak Imlarp for virus identification; Panor Srisongkram, Ming Choohong, and Piyanard Chuasuwan for antibody
measurements; Christine Kozik, Chitchai Hemachudha, Tipawan
Kungvanrattana, and Warinda Srikham for data entry and database
management; Robert Lew for statistical support; and Charles H.
Hoke, Jr., the Directors of the Bangkok Childrens and Kamphaeng
Phet Provincial Hospitals, and the Thai Ministry of Public Health
Dengue Hemorrhagic Fever Project Oversight Committee for their
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