3. Dengue Diagnostics In areas of the world where dengue disease is endemic, including Indonesia, all the
cases of acute febrile illness, presenting with nonspecific features, would have dengue viral infection included in the list of differential diagnosis. The steps in assessing a case
including prompt anamnesis, physical examination, laboratory and imaging studies. Laboratory investigations can be classified as disease monitoring laboratory test or
diagnostics tests.
7 – 10
3.1. Disease monitoring laboratory tests
Complete Blood Count CBC.White blood count in the beginning febrile phase
usually normal, and rapidly decrease as the disease progress and shows relative lymphocytosis. The presence of non specific leucopenia should raise suspicion of
possible dengue infection in appropriate setting. The rise of Hematocrit best known as Hemoconcentration as a result of plasma leakage, differentiate between dengue fever
and dengue hemorrhagic fever. Early fluid replacement or blood loss due to bleeding manifestation may mask the Hemoconcentration. It is off a great importance to obtain a
baseline Hematocrit in early febrile phase so it can be used to detect early recognition of plasma leakage. Thrombocytopenia might be the most common and prominent
laboratory feature in dengue cases. In the early febrile phase, platelet count is usually within normal range and will decrease rapidly as the disease progresses to the late
febrile phase or at defervescence. The thrombocyte may continue to remain low for the first few days of recovery phase. There is a significant negative correlation between
disease severity and platelet count but it is not predictive of bleeding.
Liver profile evaluation. Abnormal transaminates is commonly seen in dengue
infection. The elevation of AST usually greater compared to AST. The degree of transaminates usually higher in DHF compared to DF.
3.2. Diagnostic tests
Definitive diagnosis of dengue infection can only be concluded by laboratory examinations. However the Interpretation of laboratory diagnostic results shouldonly be
done in the clinical context. Laboratory confirmatory tests include detection of dengue virus and or any of its component protein NS1 antigen, virus isolation and detection of
virus genetic materials by polymerase chain reaction
– PCR or by detecting the antibody response serology response after infection Table 1.
Table 1. Dengue diagnostic and sample characteristics Clinical sample
Diagnostic method
Methodology Time
to result
Virus detection
and Its components
Acute serum 1 –
5 days of fever and necropsy
tissue Viral isolation
Mosquito or
mosquito cell
culture inoculation
One week or more
Nucleic acid detection
RT – PCR and
real time RT –
PCR 1 or 2 days
Antigen detection
NS1 Ag rapid test
Minutes NS1 Ag ELISA
1 day Immuno
– histochemistry
2 – 5 days
Serological response
Paired sera acute serum from 1
– 5 days and second
serum 15 – 21
days after Ig M or Ig G
seroconversion ELISA
HIA 1
– 2 days Neutralization
test Minimum
7 days
Serum after day 5 of fever
Ig M detection recent
infection ELISA
1 or 2 days Rapid tests
Minutes Ig G detection
Ig G ELISA HIA
1 or 2 days ELISA= enzyme-linked immunosorbent assay; HIA = haemagglutination inhibition
assay; IgG = immunoglobulin G; IgM = immunoglobulin M; NS1 Ag = Non - structural protein 1 antigen; RT
– PCR = reverse transcriptase polymerase chain reaction. Source: WHO
– TDR 2012 10 Three main aspects should be considered for an adequate dengue diagnosis:
• Virological and serological markers in relation to the time of dengue infection; • Type of diagnostic method in relation to clinical illness;
• Characteristics of the clinical samples
A. Virological and serological markers in relation to the time of dengue infection
In a primary infection viremia develops from 1 – 2 days before the onset of fever
until 4 – 5 days after. Accordingly, anti-dengue IgM specific antibodies can be detected
3−6 days after fever onset. Low levels of IgM are still detectable around one to three months after fever. In addition, the primary infection is characterized by slowly
increasing but low levels of dengue-specific IgG, becoming elevated at days 9 – 10.
Low IgG levels persist for decades, an indication of a past dengue infection. A totally different picture is observed during a secondary infection, with a rapid and higher
increase of anti-dengue specific IgG antibodies and slower and lower levels of IgM. High IgG levels remain for 30
– 40 days. A short-lasting but higher viremia level characterizes the secondary infection compared to the primary infection Figure 3.
Figure 3. Virological and serological markers of dengue infection according to time of illness
; IgG = immunoglobulin G; IgM = immunoglobulin M
B. Type of diagnostic method in relation to clinical illness
The diagnostic method to confirm an acute infection depends on the time of clinical illness: the febrile phase is coincident with the presence of viremia, some viral
components and replication products in blood; the critical and convalescent phases coincide with the development of antibodies, as summarized in Table 1.
Febrile phase day 1 to day 4
– 5 of fever, diagnostic test best using isolation of infective virus thus allow detection of serotype of dengue virus. Virus genome
detection using RT – PCR confirms the diagnosis during this phase. NS1 is a marker
of acute dengue infection. Critical and convalescent phase after day 4
– 5 of fever, specific IgM is the best marker for recent dengue infection. High levels of IgG
collected early after fever onset also suggested a recent infection
C. Characteristics of the clinical samples
Similar to other enveloped viruses, dengue virus is labile and readily inactivated at temperatures above 30°C, so care should be taken during transportation and storage
of samples. Serum samples collected during the first 4 days of fever are useful for virus, genome and dengue antigen detection, thus confirming a dengue infection.
Samples should be rapidly transported at 4°C to the laboratory and be processed as soon as possible. Sterile serum without anticoagulant is useful. If specimen delivery
cannot be performed in the first 24
–48 hours, freezing at –70°C is recommended.
The pitfalls in diagnostic test for dengue infection
Serological test for dengue have been known to cross – react with: other flaviviridae
Japanese Encephalitis, Non – Flavivirides infections malaria, leptospirosis,
toxoplasmosis, syphilis, connective tissue disease rheumatoid arthritis.
11,12,13
Secretion of the NS1 protein is a hallmark of flavivirus infection on mammalian cells and can be found in dengue infection as well as in yellow fever and West Nilevirus
infection. This antigen is present in high concentrations in the serum during early phase of the disease. The detection rate is much better in acute sera of primary
infection 75- 97.3 compared to the acute sera of secondary infection 60 70. The sensitivity of NS1 antigen detection starts to drop off from the day 4-5 of illness
and is usually undetectable in the convalescence phase.
Laboratory confirmation of a dengue case
A diagnosis of dengue infection is confirmed by the detection of the virus, the viral genome or NS1 Ag, or seroconversion of IgM or IgG from negative to positive IgMIgG
or four-fold increase in the specific antibody titre in paired sera Table 2.
Table 2. Confirmed and probable dengue diagnosis, interpretation of results and sample characteristics
Method Interpretation
Sample characteristics
Confirmed dengue
infection Viral isolation
Virus isolated Serum collected at 1
– 5 days of
fever Necropsy tissues
Genome detection
Positive RT-PCR or positive real-time
RT-PCR
Antigen detection
Positive NS1 Ag Positive
immunohistochemical Necropsy tissues
IgM seroconversion
From negative IgM to positive IgM in
paired sera Acute serum days 1
– 5 and
convalescent serum 15
–21 days after first serum
IgG seroconversion
From negative IgG to positive IgG in
paired sera or 4-fold increase IgG
levels among paired sera
Probable dengue
infection Positive IgM
Positive IgM Single serum collected
after day 5 High IgG levels High IgG levels by
ELISA or HI ≥ 1280
ELISA= enzyme-linked immunosorbent assay; HIA = haemagglutination inhibition assay; IgG = immunoglobulin G; IgM = immunoglobulin M; NS1 Ag = Non - structural
protein 1 antigen; RT – PCR = reverse transcriptase polymerase chain reaction.
Source: WHO – TDR 2012 10
4. Recommendation for Clinical Management
There is no currently available anti viral medication against the dengue virus after the host invasion. The of dengue infection management stays symptomatic and
supportive. Dengue is a dynamic disease and management issues vary according to the three phases of the clinical course. It is crucial to recognize plasma leakage and
shock at an early stage, to guard against severe organ impairment. This can only be achieved through frequent clinical and laboratory monitoring.
4.1 Clinical Management The stepwise approach for management of dengue can be used to assessing
dengue case investigation Table 3. Figure 4, Figure 5, Figure 6, Figure 7 and and Figure 6 show the summary of management decision.
Table 3. A stepwise approach to the management of dengue Step I
– Overall assessment I.1
History, including symptoms, family history and past medical history
I.2 Physical examination, including full physical and mental asessement
I.3 Investigation, including routine laboratory tests and dengue-specific laboratory
tests
Step II − Diagnosis, assessment of disease phase and severity Based on evaluations from history, physical examination +- CBC and HCT, the
clinicians should be able to determine: 1. Diagnosis of Dengue Fever suspected, probable or confirmed
2. The phase of illness febrilecriticalconvalescent or recovery 3. The hydration and hemodynamic status of patient
4. Whether the patient requires admission Step III
– Management III.1 Disease notification
III.2 Management decisions. Depending on the clinical manifestations and other
circumstances, patients may: - be sent home Group A
- be referred for in-hospital management Group B - require emergency treatment and urgent referral Group C
