1. 10.1586/ERI.12.76 895ISSN 1478-7210www.expert-reviews.com
Review
© 2012 Kin Fai Tang
Dengue is endemic throughout the tropical
world. The WHO has estimated that approxi-
mately 3 billion people live at risk of infection
each year. Infection produces a spectrum of
clinical manifestation, from mild influenza-like
illness to dengue fever (DF) or severe dengue
illness. The latter comprise of either plasma leak-
age, which leads to hypovolemic shock or dengue
shock syndrome and internal hemorrhage, or
other organ failure, including encephalo­pathy [1].
The disease is caused by dengue virus (DENV),
which belongs to the genus Flavivirus of the
Flaviviridae family [2]. DENV is a positive-sense,
single-stranded RNA virus (approximately 11 kb
in length). The genome is transcribed as a single
open reading frame encoding three structural
(C, prM and E) and seven nonstructural (NS1,
NS2A, NS2B, NS3, NS4A, NS4B and NS5)
proteins [3] that are subsequently cleaved into
individual components by proteolytic cleavage
[4]. The untranslated terminal regions at both
ends of the viral genome (3′ and 5′ untranslated
terminal regions) are important in the regula-
tion of translation and replication of the viral
genome [5].
DENV is composed of four antigenically
distinct serotypes. Infection by a specific sero-
type confers lifelong immunity against the
specific serotype but not to the remaining
three, although transient crossprotection has
been observed within 2–3 months following
acute dengue infection [6]. Secondary infection
carries a higher risk of plasma leakage that, if
not appropriately supported clinically with fluid
management, can lead to shock [7]. This asso-
ciation between secondary infection and severe
dengue is thought to be mediated by the bind-
ing of crossreactive, neutralizing antibodies at
subneutralizing concentration that enhances the
infection of monocytes and dendritic cells (DCs)
via the Fc receptors, a process termed antibody-
dependent enhancement (ADE) [8–10]. Besides
ADE, other factors that could influence severe
clinical outcome in a dengue infection include
both human host [11–14] and viral factors [15–18].
DENV is transmitted to humans primarily
by infected Aedes aegypti – the predominant epi-
demic vector – while Aedes albopictus and Aedes
polynesiensis have also caused dengue outbreaks
[19–21]. Current methods of disease prevention
rely on reducing the vector population density.
However, given the experience of countries
such as Singapore, where periodic epidemics of
dengue continue despite concerted public health
efforts to control the vector population [22], a
cost-effective vaccine remains the most viable
option for dengue prevention.
The development of a dengue vaccine has been
complicated by the concern that subneutralizing
levels of antibodies may paradoxically increase
the risk of severe dengue in the form of dengue
hemorrhagic fever (DHF) and dengue shock
syndrome through ADE [8–10]. Hence, while a
dengue vaccine was initially advocated in the
Kin Fai Tang*1
and
Eng Eong Ooi1,2
1
Program in Emerging Infectious
Disease, Duke-NUS Graduate Medical
School Singapore, 8 College Road,
169857 Singapore
2
DSO National Laboratories, Singapore
*Author for correspondence:
Tel.: +65 651 67406
Fax: +65 622 12529
kinfai.tang@duke-nus.edu.sg
Early diagnosis of dengue, the most common mosquito-borne disease globally, remains
challenging. Dengue presents initially as undifferentiated fever, with symptoms becoming
more pathognomonic in the later stages of illness. This limits the timeliness in the delivery of
appropriate supportive interventions. Laboratory tests are useful for diagnosis although the
short-lived viremia and the presence of secondary infection with one of the four heterologous
viral serotypes collectively complicate the choice and interpretation of laboratory tests. In this
article, the authors review the various approaches for diagnosis of dengue and discuss the
appropriate tests to use, including when a dengue vaccine, which is in the late stages of
development, is licensed for use. The ensuing reduced dengue prevalence could make diagnosis
for vaccine efficacy and escape-mutant monitoring even more challenging.
Diagnosis of dengue:
an update
Expert Rev. Anti Infect. Ther. 10(8), 895–907 (2012)
Keywords: dengue • diagnostics • early diagnosis • surveillance • vaccine
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2. Expert Rev. Anti Infect. Ther. 10(8), (2012)896
Review
1940s [23], it was not until 1971 that the feasibility of a dengue
vaccine in preventing DHF was studied [10,24]; and based on initial
data [25–29], a vaccine search was initiated, endorsed and discussed
by the SEARO/WHO Research Study Group and experts in the
field [24]. Despite initial optimism [30,31], more than three decades
have passed without a licensed dengue vaccine in the market.
However, current developments are promising and six tetravalent
candidate vaccines are in Phase I–III trials. Optimistically, a
licensed vaccine can be anticipated in the next 5–7 years [32–34].
Meanwhile, apart from vector control, the burden of dengue on
society can also be reduced through appropriate and timely clini-
cal interventions to prevent severe morbidity and mortality. This
relies on early and accurate diagnosis of dengue. Even when a vac-
cine or an antiviral drug becomes available, the need for accurate
diagnosis would not be diminished. Instead, the requirement for
accurate diagnosis could become more demanding, as surveillance
for dengue in vaccinated individuals would be needed to deter-
mine vaccine efficacy and for the early detection of vaccine-escape
mutants. The goal of this review is thus to determine the state of
the art in diagnosis of dengue and identify areas where improve-
ments through research are needed to prepare for the quality of
diagnostics needed in a postvaccination world.
Current status in diagnosis of dengue
Clinical diagnosis
Diagnosis of dengue starts with a clinical suspicion, prompted by
the recognition of a collection of presenting symptoms and signs. In
the early acute febrile phase of illness, dengue patients often present
with a history of sudden onset fever, which is often accompanied
by nausea, aches and pains. Unfortunately, these symptoms are not
unique to dengue and are reported with other febrile illnesses (OFI).
The onset of a maculopapular rash, retro-orbital pain, petechiae or
bleeding nose or gums are more pathognomonic of dengue and
would more probably trigger a differential diagnosis of dengue,
although these symptoms usually appear in the later stages of illness,
nearer the phase of fever defervescence, when plasma leakage occurs
[1].Their usefulness for early diagnosis would thus be more limited.
A list of the commonly reported symptoms is shown in Table 1.
As each of the individual symptoms cannot accurately differenti-
ate dengue from OFIs, an alternative approach to clinical diagnosis
is to use a permutation of a list of symptoms or signs. The WHO
guidelines for dengue are such examples [1,35]. Indeed, when applied
to prospectively recruited patients who presented with acute febrile
illness less than 72 h from fever onset, both the 1997 and 2009WHO
guidelines showed a similar sensitivity of over 95% in young adults,
albeit with poor specificities of less than 40% [12]. Consequently, the
WHO classification schemes can be useful to trigger a suspicion of
dengue. During epidemics, when the prevalence of dengue is high,
cases that fit these definitions could be treated as presumptive dengue
while awaiting other test results. However, they cannot be used for
a confirmatory diagnosis of dengue. Furthermore, caution must be
exercised in places where dengue infection occurs in older adults.
The same study showed that adults who are 56 years of age and older
had greatly reduced sensitivities, from over 95 to 73.7% and 81.6%
for the 1997 and 2009 WHO classification schemes, respectively.
In such cases, the study suggested that leukopenia in patients with
febrile illness should trigger a suspicion of dengue [12].
Besides the WHO classification schemes, others have attempted
to develop diagnostic algorithms for dengue. Tanner et al. used
a data mining approach to identify a group of symptoms and
hematologic measurements to differentiate dengue from OFIs [36].
The resultant algorithm had a sensitivity and specificity of 71.2
and 90.1%, respectively. Another comprehensive multivariable
logistic regression model was also developed and validated for
distinguishing DHF from DF, DHF from DF or OFIs, dengue
from OFIs and severe dengue from nonsevere dengue. The model
was found to have a sensitivity that ranged from 89.2 (dengue
from OFIs) to 79.6% (DHF from DF) [37]. This model also
provides a tool for probability calculation and classification of
patients based on readily available clinical and laboratory data.
However, the usefulness of such algorithms remains to be tested
in different populations with different circulating DENV strains.
An important limitation in the development of useful clinical
approaches to diagnosis of dengue is the lack of standardization
with regard to study design, diagnostic criteria and data collec-
tion [38]. While this is not surprising as these studies were per-
formed by various laboratories in different countries, it limits the
development of diagnostic classification or algorithms that can
be applied internationally. Indeed, the need for more prospective
studies to construct a valid and generalizable algorithm to guide
the differential diagnosis of dengue in endemic countries remains
urgent [38].
Laboratory diagnosis
As clinical diagnosis lacks specificity, a definitive diagnosis of
dengue infection requires laboratory confirmation. A number of
different laboratory tools are available for diagnostic use. A sum-
mary of laboratory diagnostic methods used in dengue infection
is shown in Table 2 and the approximate time from illness onset at
which these diagnostic tests should be used is shown in Figure 1.
Virus isolation
Dengue viremia can be detected from 2 to 3 days prior to the
onset of fever to up to 5.1 and 4.4 days after the onset of the
disease for primary and secondary infection, respectively [39].
During this viremic period, blood, serum or plasma samples can
be used for virus isolation.
Mosquito inoculation remains the most sensitive method for
virus isolation. The isolation rate of the four serotypes of DENV
is in the range of 71.5–84.2% [40]. Various mosquito species
have been found to be useful and sensitive in dengue isolation,
including A. aegypti, A. albopictus and Toxorhynchites splendens,
where both male and female mosquitoes are susceptible [6,41–44].
These mosquitoes are inoculated intrathoracically with serum or
plasma specimens [40–44]. Specimens collected early in the course
of illness have a greater isolation rate (85.3% before day 4 of ill-
ness) than those collected later (65.4% after day 4 of illness) [40].
Furthermore, the isolation rate in patients with primary dengue
(91.0%) was higher than those with secondary dengue (77.6%).
This observation could be due to the interference of crossreactive
Tang & Ooi

3. 897www.expert-reviews.com
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antibodies with virus isolation or a faster rate of viral clearance in
patients with secondary DENV infection [39]. Either explanation,
however, suggests that the prevalence of primary or secondary
DENV infection may influence the overall virus isolation [40,45].
Mouse brain inoculation has also been used to isolate and
amplify DENV. Generally, unweaned mice (2–4 days of age)
are inoculated intracerebrally with serum or plasma specimens
and observed daily. Moribund mice are then sacrificed to harvest
the isolate [46,47].
Both mosquito and mouse brain inoculation techniques are
not routinely used in day-to-day diagnosis owing to their highly
specialized technical, safety and facility requirements as well as
high maintenance costs. Instead, virus isolation using cell lines
is more widely used. The most commonly used cell line for
DENV isolation is C6/36, which was derived from A. albopictus.
Alternatively, mammalian cell lines such as Vero, LLC-MK2 or
BHK-21 could also be used, although these offer lower sensitiv-
ity than C6/36 [41,42,48–53]. Besides diagnosis, virus isolation
offers the advantage of providing a virus isolate that may be
characterized during subsequent in vitro studies, such as genome
­sequencing, virus neutralization and infection studies.
A successful isolation of DENV on mosquito or cell culture
can be confirmed and serotyped by an immunofluorescence
assay using DENV- and serotype-specific monoclonal anti­bodies,
respectively [41,53]. Virus isolation is highly specific and has a
theoretical detection limit of a single viable virus, although in
practice, the sensitivity is only approximately 40.5% in cell line-
based virus isolation [54]. It also requires highly trained operators,
a dependence on sample integrity and a short viremia period, thus
providing a narrow window of opportunity from illness onset [54].
Therefore, despite its advantages, this approach is not widely used
in routine diagnostic laboratories.
Viral RNA detection
Reverse transcriptase PCR (RT-PCR) detection of dengue viral
RNA extracted from blood, serum or plasma provides a rapid,
sensitive and specific method for dengue infection confirmation.
Various primers and protocols have been developed, validated and
used in conventional RT-PCR [1,54–61] and real-time RT-PCR,
either using SYBR®
Green as a fluorescent detection marker or
labeled oligonucleotide probes [58,59,62–79]. A technique using a
single reaction mixture at a constant temperature (nucleic acid
Table 1. Symptoms differentiating dengue infection from other febrile illnesses.
Symptoms Den|OFIs p-value Children (<15 years)|adults
(>15 years)
p-value Ref.
Nausea 50.0|28.9%
68.0|49.0%†
51.3|30.5%
<0.00001
<0.05
<0.001
50.2|76.4% <0.001 [12,103,145,146]
Vomitting 16.4|8.4%
16.2|8.5%
57.0–64.0|31.0–46.0%†
70.0|52.0%†
<0.00001
0.03
<0.01
<0.05
50.2|76.4% <0.001 [12,103,145–148]
Retro-orbital pain 26.0|15.9%
26.6|13.5%
10.01§
<0.00001
0.003
0.001
8.7|29.1% <0.001 [12,103,146,148]
Aches/pains 1.4§
<0.0001 20.3|36.4% 0.012 [12,146]
Rash 11.2–41.2|3.0–6.4% <0.003/0.007 NA NA [12,103,134,149]
Tourniquet test positive 34.0|19.0%
42.0|5.0%†
43.0–65.0|21.0–39.0%
1.86§
0.02
<0.01
<0.1
<0.001
NA NA [12,103,134,149]
Leukopenia 3.8 × 103
|7.3 × 103
/µl
4.5 × 103
|8.1 × 103
/μl
<4.5 × 103
/μl: 72.1|11.5%
<0.0001
<0.1
<0.001
NA NA [12,37,103,145]
Thrombocytopenia
(platelet/mm3
)
16|4% (≤100,000)†
16|82%‡
(≤100,000)
66|95%‡
(≤100,000)
14.9|1.5% (≤100,000)
32,000|96,500
163,500|239,000
70,000|104,000¶
NA
NA
<0.01
<0.001
<0.001
<0.0001
NA
NA NA [12,103,145,147,150]
†
Studies perfomed in children younger than 15 years.
‡
Dengue and severe dengue comparison, performed in children younger than 15 years.
§
Risk ratio.
¶
Average.
Den: Confirmed dengue cases; NA: Not applicable; OFI: Other febrile illness.
Diagnosis of dengue: an update

4. Expert Rev. Anti Infect. Ther. 10(8), (2012)898
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sequence-based amplification [NASBA]) [80] was found to be
highly sensitive (98.5%) and specific (100%) [81,82]. NASBA may
be highly useful and applicable during outbreak field diagnosis
where thermocyclers are not readily available.
Sensitivity of conventional RT-PCR ranges from 48.4 to
98.2% and has a detection limit of 1–50 plaque forming units
(PFUs) [54–56,59]. These assays employ primers that bind to
known conserved regions of the DENV genome to avoid false
negative findings due to spontaneous mutations expected in
the replication of the RNA viral genome. The use of in silico
methods to develop a cocktail of primers that bind to almost
all DENV with known sequences has also been explored [58],
although validation in a clinical setting remains to be carried
out. The sensitivity of RT-PCR is also highly dependent on the
short window of opportunity that coincides with the viremic
period, which can last up to 8 days from illness onset (Figure 1).
However, RT-PCR is rarely positive in a case of dengue after
6 days from illness onset [1].
Fluorescence-based real-time RT-PCR has a better reported sen-
sitivity (58.9–100%) and detection limit (0.1–3.0 PFUs) owing to
the sensitivity of the fluorescence detector within the thermocycler
[59,62–67,71,74,75,79]. Multiplex RT-PCRs that differentiate DENV
serotypes in a single assay have also been developed [56,59,65,69,76].
The experience with NASBA is more limited compared with
RT-PCR, although a study has shown that
it can be as sensitive as RT-PCR, with a
detection limit of <25 PFUs/ml [81]. RNA
extraction from whole blood may be more
sensitive (90.0%) than serum or plasma
(62.0%) in the same pool of samples [78].
Besides blood samples, RT-PCR can also
be used to detect DENV RNA in tissues,
including formalin-fixed specimens [82].
Although RT-PCR usually requires exper-
tise in molecular techniques and expensive
equipment [83], modified protocols using
fast-ramping thermocyclers can be used
in conjunction with newly trained opera-
tors during emergency settings, such as in
differentiating dengue from SARS during
an outbreak [62], provided a strict standard
operating procedure is followed.
Dengue viral RNA can also be detected
in urine [68,72] and saliva [72] samples using
real-time RT-PCR. In urine, samples
–2 –1 0
Disease
onset
3
Time (Days)
1 2 4 5 6 7 8 9 10 11 20 30 40 60 90
lgM
lgM
lgG
lgG
2* dengue
1* dengue
>90
Viremia
NS1
Virus isolation
Viral RNA detection
Figure 1. Approximate window of detection for dengue diagnostics.
NS1: Non-structural protein 1.
Data taken from [1].
Table 2. Laboratory diagnostics for dengue: sensitivity and specificity.
Category Technique Parameters Ref.
Sensitivity (%) Specificity (%) Detection limit
Viral detection Virus isolation (mosquitoes) 71.5–84.2 100 NA [6,40–42,44]
Virus isolation (mouse intrecerebral inoculation) NA NA NA [46,47]
Virus isolation (cell culture) 40.5 100 ≥1 viable virus [54]
Viral RNA RT-PCR (conventional) 48.4–100 100 1–50 PFUs [54–57]
Viral RNA RT-PCR (real-time detection) 58.9–100 100 0.1–3.0 PFUs [54,57,63–67,79]
Viral RNA RT-PCR (NASBA) 98.5 100 <25 PFUs/ml [81]
Viral antigen detection (NS1 detection) 54.2–93.4 92.5–100 0.2 ng/ml†
[79,91–103,110]
Antibody detection IgM detection 61.5–100 52.0–100 NA [54,102,115,116]
IgG detection 46.3–99.0 80.0–100 NA
Rapid IgM detection (strips) 20.5–97.7 76.6–90.6 NA [115]
Antigen/antibody
combined detection
NS1 and IgM 89.9–92.9 75.0–100 NA [91,100–102]
NS1 and IgM/IgG 93.0 100 NA [101,151]
NA: Not applicable; NASBA: Nucleic acid sequence-based amplification; PFU: Plaque forming unit; RT-PCR: Reverse transcriptase PCR.
†
Data taken from [110].
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5. 899www.expert-reviews.com
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collected between day 6 and day 16 after illness onset were
found to have higher rates of detection (50–80%) compared
with day 1 to 3 samples (25–50%) [68]. RT-PCR for DENV
in urine may thus extend the window of opportunity for viral
RNA detection compared with blood specimens (up to day 8).
However, the level of viral RNA in urine and saliva samples is low
(1 × 101
–5 × 101
PFUs/ml) compared with the corresponding serum
samples (7.9 × 102
–1.9 × 105
PFUs/ml) [72].
Antigen detection
Dengue NS1 is a highly conserved glycoprotein essential for
DENV viability and is secreted from infected cells as a soluble
hexamer [84,85]. Serum or plasma DENV NS1 level has been found
to correlate with viremia titer and disease severity [86–88]. It can
be found in the peripheral blood circulation for up to 9 days
from illness onset [89–91], but can persist for up to 18 days from
illness onset in some cases [92]. NS1 detection thus offers a larger
window of opportunity for diagnosis of dengue compared with
virus isolation, RT-PCR or NASBA [68,72]. Commercially available
NS1 capture-based detection kits with sensitivities that ranged
from 54.2 to 93.4% have been comprehensively evaluated (Table 2)
[66,79,91,93–103] and found to be able to confirm dengue infection
in serum specimens that were RT-PCR negative and secondary
dengue infection [97]. However, NS1 detection is less sensitive in
secondary dengue infection (67.1–77.3%) compared with primary
dengue cases (94.7–98.3%) [93,94,96,103], probably owing to the
presence of crossreactive anti-NS1 antibodies that impedes the
detection of free NS1 proteins in the serum or plasma [86,89].
Anti-NS1 antibodies can also be used to detect infection in
other sample sources, such as tissues, including liver, lung and
kidney [104], through immunohistochemistry. This could be use-
ful in postmortem studies. Although highly conserved, serotype-
specific NS1 monoclonal antibodies have been raised and applied
for NS1-based dengue serotype identification assays [105–109]. A
study by Puttikhunt et al. has shown an overall sensitivity of 76.5%
and specificity of 100% for diagnosis of dengue while having sero­
typing sensitivities of 100% for DENV 1, 3 and 4 and 82.4% for
DENV2 [109]. However, the sensitivity of these tests may differ
with different strains of DENV as the magnitude of NS1 secretion
appears to be strain dependent [110].
Antibody detection (IgM & IgG)
Detection of antidengue antibodies (IgM and IgG) is the most
widely used test in diagnosis of dengue [111].These kits are either in
the form of Ig capture or direct Ig detection and are configured to
detect IgM, IgG or both simultaneously [102,112–115].There are two
versions of these tests: ELISA or strip format (rapid test). While
ELISA provides greater sensitivity, the strip format is amenable
for bedside use [116].
Antibody response in the form of antidengue IgM can be
detected as early as 3–5 days after illness onset. Levels of IgM
continue to increase for approximately 2 weeks thereafter and may
persist for approximately 179 and 139 days following primary and
secondary infection, respectively [117]. Thus, while a single IgM
raises the likelihood that a febrile patient has dengue, a definitive
diagnosis may require the use of paired sera to demonstrate rising
IgM titers.
A multinational and multicenter study of ten IgM kits has
concluded that ELISA-based detection kits have higher sensi-
tivities (61.5–99.0%) compared with the rapid test formats
(20.5–97.7%). The specificities are in the range of 79.9–97.8%
and 76.6–90.6% for ELISA and rapid tests, respectively [115].
Other evaluation studies have also reported similar sensitivities
and specificities [62,102,116]. The wide ranges of these values are
probably due to the timing of sample collection [118].
Early antidengue IgM response (<2 months) has been found to
be crossreactive to all four DENV serotypes [119] and other flavi­
viruses [115]. Hence, epidemiological information on the preva-
lence of other flaviviruses would be useful to guide the interpre-
tation of a positive IgM finding. False positives have also been
observed in patients with previous dengue or malaria infection
[116]. However, more efficient algorithms can be developed to
mitigate this ­problem, as shown by Prince et al. [120].
During primary infection, IgG can only be detected after
10 days from illness onset, making it less useful for early diagno-
sis. However, the rapid increase of IgG levels during secondary
infection (as early as day 4 from illness onset) [1] can be suggestive
of dengue when the ratio of IgM and IgG is used [62,102,115–117].
Dengue neutralizing antibody detection
Neutralizing antibodies inhibit DENV infection and can thus
provide greater specificity in distinguishing antibodies to DENV
from other crossreactive flavivirus antibodies [121,122]. These anti-
bodies can be measured by using plaque reduction neutralizing
tests (PRNTs), first developed by Russell et al. [122] based on the
protocol from Dulbecco et al. [123]. To date, PRNT remains the
most widely used assay for immunity studies [124,125]. However, it
is labor intensive, time consuming and has low throughput [124],
and is therefore not routinely used in dengue diagnostics.
New tests such as the ELISA-based microneutralization test
(ELISA-MN) [126], the fluorescent antibody cell sorter-based
Dendritic Cell-Specific Intercellular adhesion molecule-3-Grab-
bing Non-integrin expressor DC assay [127] and the enzyme-linked
immuno­sorbent spot microneutralization assay [128] have been
developed to overcome the limitations associated with PRNT.
These new tests have been separately validated, compared and eval-
uated [124,126–130] against PRNT and found to have good agreement
(false-positive rate <10%) in cases with primary DENV infection
[124,130]. However, Putnak et al. reported poor agreement among
the tests in association with vaccination or secondary infection
[124]. This result could have been influenced by the use of different
cell lines or different strains of DENV [129]. Others have suggested
the use of Fcγ receptor (FcγR)-positive cells for these assays since
DENV infects monocytes and DCs that express such receptors
[131,132]. The use of such cells may also provide information on
whether the antibodies were able to neutralize DENV intra­
cellularly or whether neutralization was only mediated through
the coligation of FcγRIIB, which inhibits FcγR-mediated phago-
cytosis and hence DENV entry into monocytes [133]. A limited
observation suggests that this could provide greater specificity on
Diagnosis of dengue: an update

6. Expert Rev. Anti Infect. Ther. 10(8), (2012)900
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the DENV serotype responsible for the infection [133]. However,
detailed validation is required for all of these assays before they
can be used clinically.
Combined antigen/antibody detection
Given the dynamic nature of the NS1 antigen, antidengue IgM
and IgG antibody levels during the course of acute illness, efforts
have been made to combine all three tests into a single reaction
for ease of use. Some of these have been evaluated and shown to
have promising diagnostic sensitivity (89–93%) and specificity
(75.0–100%) [91,100–102,134].
Advances in rapid diagnostic tools
While rapid bedside diagnosis formats are available for antigen
or antibody detection or both simultaneously, the sensitivities
and specificities of the available tests have been uniformly lower
than the equivalent laboratory-based assays. A complete review of
these assays is provided elsewhere [102,118]. These limitations may
be due to the use of the lateral flow dipstick approach. The new
lab-on-a-chip platform could offer a way to improve the perfor-
mance of these bedside diagnostic tools [135–137]. This platform
makes use of a number of new technologies; some in combina-
tion, such as microfluidics [135–137] and grating couplers [137] to
improve multiplexing, accommodate better mixing of reagents
with test samples as well as achieving greater sensitivity for detect-
ing positive signals [138]. This platform could feature prominently
in dengue diagnostics in the coming years.
Disease prognostication
Progression of mild dengue infection to severe dengue
(DHF/dengue shock syndrome) is difficult to predict owing to an
incomplete understanding of disease pathogenesis. Symptoms and
signs of severe dengue have a sudden onset at the time of deferves-
cence [1,19]. Careful monitoring of hematocrit as well as signs of
circulatory failure or internal hemorrhage needs to be carried out
for at least 2 days after fever defervescence. Specifically, patients
should be observed for signs such as severe abdominal pain, passage
of black stools, bleeding into the skin, nose or gums, sweating or
cold skin, which could indicate the devel-
opment of DHF [7]. Depending on disease
progression, should DHF, occurs, oral rehy-
dration therapy is sufficient for milder DHF
while intravenous fluid therapy is suggested
for more severe manifestations, and blood
­transfusion is suggested for ­critical cases [7].
However, hospitalization for close mon-
itoring of all patients in dengue-endemic
countries is often not feasible, particularly
during outbreaks, as it stresses the lim-
ited medical healthcare resources. Under
such circumstances, an ability to predict
the development of severe dengue at the
early stages of illness could thus be use-
ful for triaging patients. Various clinical
markers have been proposed as warning
signs of severe dengue progression, as
shown in Table 3. How well these clinical
symptoms/signs perform in predicting the
onset of severe dengue remains to be fully
determined.
Besides monitoring individual symptoms
or signs, several groups have also evaluated
the usefulness of combining these into an
algorithm for predicting severe dengue. Lee
et al. explored the use of a probability equa-
tion that combines four simple clinical lab-
oratory observations, including bleeding,
lymphocyte proportion, increased serum
urea and low total serum protein [139]. They
reported a sensitivity of 100% and speci-
ficity of 46%. They estimated that 43.9%
of the mild dengue cases could have been
prevented from hospitalization in 2004.
The authors followed up this study with a
Table 3. Warning signs and symptoms leading to potential severe
dengue (dengue hemorrhagic fever/dengue shock syndrome).
Warning signs for severe disease progression
Signs Den|OFIs p-value Ref.
Abdominal pain & tenderness 63.3|22.6% <0.05 [150,152]
78.0|22.0%†
0.03
Persistent vomiting NA NA [153]
Clinical fluid accumulation 46.4|7.1% 0.002 [154]
Mucosal bleed/ spontaneous bleeding 84.1|65.9% 0.008 [150,153]
26.6|10.4% 0.05
Lethargy, restlessness 12.7|2.2% 0.05 [150,152]
54.0|20.0%†
0.002
Liver enlargement >2 cm 91.6|72.4%‡
0.01 [146]
Severe dengue (DHF/DSS)
Symptoms Mild|severe p-value Ref.
Severe plasma leakage leading to
shock and fluid accumulation with
respiratory distress
92.9–100% in severe den NA [154]
Severe bleeding (evaluated by clinician) 10.0–35.7% in severe den <0.01 [139,154]
Gums: 5.7–6.0|65.0–67.8%
Nose: 0.9|12.0–16.9%
Severe organ involvement AST: 1293|196 IU/l 0.015 [37,145,153,155]
Liver: increased AST/ALT ALT: 309|132 IU/l 0.075
CNS: impaired consciousness AST: 76|12 U/l <0.01
Heart and other organs ALT: 30|20 U/l <0.01
Thrombocytopenia ≤100,000/mm3
16|82% NA [145]
†
Dengue and severe dengue comparison, performed in children younger than 15 years of age.
‡
Children (younger than 15 years of age) versus adults (older than 15 years of age).
ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; Den: Confirmed dengue cases;
DHF: Dengue hemorrhagic fever; DSS: Dengue shock syndrome; IU/l: International units per liter;
NA: Not applicable; OFI: Other febrile illness.
Tang & Ooi

7. 901www.expert-reviews.com
Review
prospective validation of their equation in the same hospital and
found similar levels of accuracy [140].
Another algorithm using platelet count, crossover threshold
of PCR-positive results (viremia in blood) and pre-existing anti­
dengue IgG (secondary infection) measured during the first 72 h
of illness was shown to predict hospitalization with a sensitivity
and specificity of 78.2 and 80.2%, respectively [36]. Likewise,
as discussed earlier, the algorithm that distinguishes DHF from
dengue infection is able to achieve a sensitivity of 79.6% [37]. The
ability to calculate the probability of development of severe den-
gue based on routinely performed clinical tests could also be use-
ful to guide prognostication [37]. However, further prospective
clinical studies are needed to validate their usefulness.
Quality assurance
While the authors have reviewed the sensitivities and specificities
of the various tools for diagnosis of dengue, how these tests actu-
ally perform can be affected by a number of variables that differ
from laboratory to laboratory, or from region to region. Thus,
quality assurance programs should be instituted in all diagnos-
tic and reference laboratories that offer services in diagnosis of
dengue. This ensures that the tests perform at the expected levels
in different laboratories and in different hands. Details on such
quality assurance programs have been reviewed elsewhere [141].
Diagnosis of dengue in a vaccinated population
While an effective and safe vaccine against dengue is anticipated,
its introduction could also provide fresh challenges for diagnosis
of dengue. Even though the goal of vacci-
nation is to eliminate dengue cases entirely,
there are currently no data that indicate
that a complete elimination of DENV is
feasible with vaccination programs. On the
contrary, there remains a concern that the
antibodies generated by vaccination may
enhance dengue, particularly when anti-
body levels wane in the years following vac-
cination. Furthermore, vaccination may not
prevent infection against all strains [142] or
may drive the emergence of vaccine-escape
mutants [143], as encountered with other
infections, such as hepatitis B [144]. A com-
prehensive surveillance of dengue among
cases of febrile illness would thus be needed
to determine the true efficacy of ­vaccination
and to monitor for ­vaccine failure.
Epidemiologically, vaccination would
reduce DENV transmission and hence the
prevalence of dengue. Under such circum-
stances, diagnostic approaches or tests with
high sensitivity but poor specificity would
result in a high false-positive rate. However,
a low sensitivity could lead to false-negative
findings, which could result in an inability
to detect the emergence of vaccine failure
or escape mutants early enough to trigger the necessary public
health responses.
Given these requirements, clinical diagnosis using symptoms,
signs and standard routine hematological or biochemical tests is
unlikely to provide sufficient specificity (Figure 1). Furthermore,
vaccinated individuals may also present with a milder illness
than classical dengue infection, making approaches such as the
use of the WHO dengue classification schemes less sensitive.
Diagnosis of acute DENV infection must thus rely even more
on the laboratory.
Serologically, DENV infection in vaccinated individuals would
also resemble that of a secondary infection, where a rise in IgM
titers is not a consistent feature but a rapid rise in IgG titers or
the ratio of IgM and IgG could be suggestive of acute DENV
infection [117]. In this respect, collection of a convalescent serum
sample to demonstrate rising antibody titers would be very use-
ful in interpreting these serological tests. Caution will need to be
exercised in places where another flavivirus, such as West Nile
or Japanese encephalitis virus, circulates. Overall, however, sero-
logical approaches will probably lack the specificity required for
a definitive diagnosis of dengue in the low prevalence setting
expected in vaccinated populations (Figure 2).
Detection of DENV or components of DENV are likely to
provide the necessary sensitivity and specificity needed (Figure 2).
While NS1 antigen detection is easy to use and is suited to point-
of-care diagnosis, the presence of vaccine-induced antidengue IgG
antibodies, as with secondary DENV infection, could lower the
overall sensitivity of this test. Likewise, while virus isolation may be
0.8
1.00
0.95
0.90
0.85
0.6
0.4
PPV
NPV
0.2
0.0
0 10 20 30 0 10 20
WHO (<56 yrs)
WHO (≥56 yrs)
PCR
NS1
lgM (ELISA)
lgM (Rapid)
30
Prevalence Prevalence
Figure 2. Positive- and negative- predictive values of the various diagnostic
approaches for dengue at different rates of prevalence. Results were generated
from median values of sensitivity and specificity presented in Table 2 for PCR
(conventional and real time), IgM (ELISA), IgM (Rapid) and NS1 detection, respectively.
A specificity of 96% was assumed for conventional PCR and real-time PCR as most
studies have limited population sizes. Diagnosis using the WHO 2009 classification was
used to represent clinical diagnosis. Data for WHO criteria were obtained from Low et al.
[12] and separated into two age groups (<56 and ≥56 years).
NPV: Negative-predictive value; NS1: Non-structural protein 1; PPV: Positive-predictive value.
Diagnosis of dengue: an update

8. Expert Rev. Anti Infect. Ther. 10(8), (2012)902
Review
highly specific, it lacks sufficient sensitivity, especially since in most
places, a suitable insectary for mosquito inoculation is not likely to
be available and laboratories will have to rely on cell cultures.
However, virus isolation will not be redundant and would need
to be done in all RT-PCR-positive specimens, as isolation of
vaccine-escape mutants would be needed to characterize these
viruses. Such information could be useful in updating vaccine
composition through the development or selection of appropri-
ate vaccine strains or even updating the primers and probes used
in ­molecular diagnostic assays [143].
Nucleic acid detection offers the highest sensitivity and speci-
ficity, and would thus be the most appropriate approach for acute
diagnosis of dengue in vaccinated populations with low disease
prevalence (Figure 2). Emphasis should be on those assays that
have been carefully validated in different laboratories serving
different populations. The availability of panels of standard-
ized positive and negative controls, along with an internation-
ally coordinated quality assurance program, would be needed
to ensure consistency in the performance of the diagnostic
assays. Presently, such a molecular diagnostic assay is lacking.
RT-PCR method used in different laboratories differ in terms
of primers/probes, enzymes and buffers as well as cycling con-
ditions. The method of detection of the RT-PCR amplicon,
whether as an end point or real-time assay, is also likely to be
different, as with the method of viral RNA extraction from clini-
cal specimens. These limitations need to be addressed urgently
if we are to be prepared for diagnosis of dengue and surveillance
in a postvaccination world.
Expert commentary
DENV and its mosquito vectors have expanded geographically
throughout the tropical world and are now encroaching into
subtropical regions. These trends make dengue a global health
concern. In the absence of either a licensed vaccine or antiviral
drug, reduction of the disease burden relies on early clinical
recognition of dengue and the timely initiation of supportive
therapy. As differentiation between dengue and other causes of
febrile illnesses is difficult based on presenting symptoms and
signs, laboratory tests are needed for a confirmatory diagnosis.
This review summarizes the current knowledge on clinical as
well as laboratory diagnosis of dengue. It reveals that clinical
approaches generally have high sensitivities but poor specificities
and discusses the various decision algorithms that have been
designed to improve the specificity of clinical diagnosis. For
confirmatory diagnosis, a range of laboratory tools are avail-
able and the main consideration on which tool to use is the
time from illness onset. A central theme of this review is the
need for a systematic validation of the performance of both the
decision algorithms and laboratory assays in different popula-
tions and diagnostic laboratory settings, respectively. This need
for quality-assured standardized performance could, paradoxi-
cally, become more acute when a dengue vaccine or antiviral
drug becomes available. The consequent reduction in dengue
prevalence necessitates the use of the most sensitive and specific
method to derive useful positive and negative predictive val-
ues to support clinical decisions in treatment and public health
responses.
Five-year view
We speculate that a dengue vaccine will be near licensing in
5 years and that potential antiviral drugs against dengue will
also enter late stages of clinical trials. The implementation of
either countermeasure against dengue would shift the emphasis
of diagnosis of dengue from serological to virological. Tools that
detect either the viral genome or antigen, particularly at the bed-
side, would gain favor. These tools are better able to distinguish
dengue from other flaviviral infections and are also useful in
the early phases of illness, when initiation of antiviral therapy
would probably exert its maximal effect. Furthermore, definitive
diagnosis of dengue in vaccinated populations would become
even more important as it could herald waning immunity or
emergence of vaccine-escape mutants; either scenario would
trigger a public health emergency. Hence, the need for improve-
ments to existing approaches for the diagnosis of dengue would
not be diminished with the advent of either vaccination or anti-
viral drug therapy, but rather the demand for tests that achieve
near-perfect sensitivity and specificity will increase in the next
5 years.
‍Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
Key issues
• Clinical diagnosis using the 1997–2009 WHO dengue classification schemes has high sensitivity but lacks specificity.
• Decision algorithms for diagnosis have been proposed but lack prospective validation.
• Choice of diagnostic assays should be guided by the time from illness onset.
• The presence of pre-existing antibodies from a previous heterologous dengue virus infection or a previous flavivirus infection can
affect the sensitivity or specificity of many diagnostic assays.
• Postvaccination surveillance would face the same challenges for diagnostics as currently encountered with secondary dengue infection.
• Despite the above, diagnosis of dengue in vaccinated individuals is critical for the surveillance of vaccine failure and escape mutants.
• Diagnostic assays with high sensitivity and specificity will be in particular demand in the low dengue prevalence setting following
vaccination.
Tang & Ooi

9. 903www.expert-reviews.com
Review
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