DENGUE

Christina Amalia
Moderator: dr. Anik Widijanti, SpPK(K)
 INTRODUCTION
Dengue fever (DF) is the fastest emerging arboviral
infection spread by Aedes mosquitoes with major
public health consequences
Up to 2.5 billion people globally live under the threat
of DF and its severe formsdengue haemorrhagic
fever (DHF) or dengue shock syndrome (DSS)
About 2.5% of those affected with dengue die of the
disease

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 VIRUS
Dengue viruses are members of the genus Flavivirus
Small (50 nm) viruses contain single-strand RNA as
genome

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 VIRUS
4 virus serotypes: DENV-1, DENV-2, DENV-3 and
DENV-4
- Infection with any one serotype confers lifelong
immunity to that virus serotype
- Secondary infection with another serotype or
multiple infections with different serotypes leads
to severe form of dengue (DHF/DSS)

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 VECTORS
Two most important vectors of dengue:

Aedes aegypti Aedes albopictus

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 HOST
Humans become dead-ends for dengue virus
transmission
Individual risk factors contribute to more severe disease:
Age (infants and the elderly)
Obesity
Pregnant women
Peptic ulcer disease
Hemolytic diseases
Chronic disease (diabetes mellitus, chronic renal
failure, etc)
Steroid or NSAID treatment
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PATHOGENESIS

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PATHOGENESIS

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 MANIFESTATION
Manifestations of dengue virus infection

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 MANIFESTATION
The course of dengue illness Febrile
Dehydration
High fever may cause
neurological
disturbances and
febriles eizures in
young children
Critical
Shock from plasma
leakage
Severe haemorrhage
Organ impairment
Recovery
Hypervolemia
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 MANIFESTATION
WHO classification of dengue infections & grading of severity of
DHF (2011)
DF/DHF Signs and Symptoms Laboratory
DF Fever with 2 of the following: Leucopenia
(WBC 5000
Headache cells/mm3)
Retro-orbital pain Thrombocytopenia
Myalgia (Platelet count <150
Arthtralgia/bone pain 000 cells/mm3)
Rash Rising haematocrit
Haemorrhagic (5% 10%)
manifestations No evidence of plasma
No evidence of plasma loss
leakage
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 MANIFESTATION
WHO classification of dengue infections & grading of severity of
DHF (2011)
DF/DHF Grade Signs and Symptoms Laboratory
DHF I Fever
Positive torniquet test
Evidence of plasma leakage
II Grade I + spontaneous bleeding
III* Grade I or II + circulatory failure: Thrombocytopenia
<100.000 cells/mm3
weak pulse HCT rise 20%
narrow pulse pressure
hypotension
restlessness
IV* Grade III + profound shock with
undetectable BP and pulse
*DHF grade III and IV are DSS
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 MANIFESTATION
DF with haemorrhage can occur in association with
underlying disease such as peptic ulcers, severe
thrombocytopenia and trauma
DHF is not a continuum of DF
Expanded dengue syndrome (unusual or atypical
manifestations) could be explained as complications
of severe profound shock or associated with
underlying host conditions/diseases or coinfections

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 MANIFESTATION
Unusual or atypical manifestations of dengue

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 MANIFESTATION
Unusual or atypical manifestations of dengue

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 DIAGNOSIS
Dengue fever

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 DIAGNOSIS
Dengue fever

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 DIAGNOSIS
Dengue hemorrhagic fever

m If all the four criteria are met, the sensitivity and specificity is 62% and 92% respectively
n If fever and significant plasma leakage are documented, a clinical diagnosis of DHF is most likely even if there is no
bleeding or thrombocytopenia
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 DIAGNOSIS
Dengue shock syndrome

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 DIFFERENTIAL DIAGNOSIS
Arboviruses Chikungunya

Other viral diseases Measles

Rubella
Influenza
Hepatitis A
Hantavirus
Enteroviruses
Epstein barr virus (EBV)
Bacterial diseases Meningococcaemia
Leptospirosis
Typhoid
Melioidosis
Rickettsial diseases
scarlet fever
Parasitic diseases Malaria

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LABORATORY DIAGNOSIS
 LABORATORY DIAGNOSIS
Diagnostic methods for detection of dengue infection

Virus isolation

Viral nucleic acid detection

Viral antigen detection

Immunological response based test

Analysis for hematological parameters

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 LABORATORY DIAGNOSIS
Diagnostic tests and phases of disease
During the early
stages of the disease
(up to 6 days of onset
of illness):
- virus isolation
- viral nucleic acid
- viral antigen
detection
At the end of the
acute phase of
infection:
- immunological tests
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 LABORATORY DIAGNOSIS
Collection, storage and shipment requirements of specimens

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 DENGUE VIRUS ISOLATION
Suitable specimens:
- acute phase serum
- plasma or washed buffy coat from the patient
- autopsy tissues from fatal cases (liver, spleen,
lymph nodes, thymus)
- mosquitoes collected from the affected areas
Tissues and pooled mosquitoes are triturated or
sonicated prior to inoculation

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 DENGUE VIRUS ISOLATION
Inoculation of serum/plasma into mosquitoes the
most sensitive
Mosquito cell culture the most cost-effective
method for routine virological surveillance

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 DENGUE VIRUS ISOLATION
Dengue virus isolation method

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VIRAL NUCLEIC ACID DETECTION
All nucleic acid detection assays involve 3 basic steps:

Nucleic acid Detection of

Nucleic acid
extraction & the amplified
amplification
purification product

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VIRAL NUCLEIC ACID DETECTION
Dengue viral nucleic acid detection method

Nested using universal dengue primers targeting the

RT-PCR C/prM region of the viral genome for an initial
reverse transcription and amplification step,
followed by a nested PCR amplification that is
serotype-specific

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 VIRAL NUCLEIC ACID DETECTION
Dengue viral nucleic acid detection method
One- A combination of the 4 serotype-specific
step oligonucleotide primers is used in a single reaction
multiple step in order to identify the serotype
x RT- The products of these reactions are separated by
PCR electrophoresis on an agarose gel
The amplification products are visualized as bands
of different molecular weights after staining the
gel using ethidium bromide dye, and compared
with standard molecular weight markers
In this assay, dengue serotypes are identified by
the size of their bands
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VIRAL NUCLEIC ACID DETECTION
Dengue viral nucleic acid detection method

Real-time A one-step assay system using primer pairs and

RT-PCR probes that are specific to each dengue
serotype
The use of a fluorescent probe enables the
detection of the reaction products in real time,
in a specialized PCR machine, without the need
for electrophoresis
Assays are either singleplex or multiplex

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 VIRAL NUCLEIC ACID DETECTION
Dengue viral nucleic acid detection method

Isothermal Does not require thermal cycling

amplificati instrumentation
on method The initial stage is a reverse transcription in
which the single-stranded RNA target is copied
into a double-stranded DNA molecule that
serves as template for RNA transcription
Amplified RNA is detected either by
electrochemiluminescence or in real time with
fluorescent-labelled molecular beacon probes

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 VIRAL ANTIGEN DETECTION
Non-structural protein 1 (NS-1)
A glycoprotein produced by all flaviviruses and is
essential for replication and viability of the virus
The protein is secreted by mammalian cells but not
by insect cells
NS1 antigen appears as early as Day 1 after the onset
of the fever and declines to undetectable levels by 5
6 days

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 VIRAL ANTIGEN DETECTION
Non-structural protein 1 (NS-1)

Can be detected in both patients with primary and

secondary dengue infections

Commercial kits for the detection of NS1 antigens do

not differentiate between the serotypes

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 VIRAL ANTIGEN DETECTION
Immunochromatographic (ICT)
Principle

Positive

Gold Conjugates : Mouse monoclonal anti-dengue

NS1gold colloid
Test: Mouse monoclonal anti-dengue NS1
Negative
Control: Goat anti-mouse IgG

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 VIRAL ANTIGEN DETECTION
Immunochromatographic (ICT)
Procedure & interpretation (Focus Dengue Dx NS1
antigen)

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 VIRAL ANTIGEN DETECTION
Enzyme-linked immunosorbent assay (ELISA)
Principle
1.Serum dengue NS1 antigen, when present, binds to
anti-NS1 antibodies attached to the polystyrene
surface of the Assay Plate
2.Residual serum is removed from the Assay Plate by
washing
3.HRP Conjugated Anti-NS1 MAb is added to the
Assay Plate

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 VIRAL ANTIGEN DETECTION
4. After incubation, the microwells are washed and a
colourless substrate system (TMB/H2O2) is added
5. The substrate is hydrolysed by the enzyme and the
chromogen changes to a blue colour
6. After stopping the reaction with acid, the TMB
turns yellow
7. Colour development is indicative of the presence
of dengue NS1 antigen in the test sample

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 VIRAL ANTIGEN DETECTION
Enzyme-linked immunosorbent assay (ELISA)
Procedure (Panbio Dengue Early ELISA)

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 VIRAL ANTIGEN DETECTION
Enzyme-linked immunosorbent assay (ELISA)
Interpretation (Panbio Dengue Early ELISA)

Index Result Interpretation

<0.9 Negative No detectable dengue NS1 antigen

The result does not rule out dengue infection
An additional sample should be tested with serology in 7-
14 days
0.9 1.1 Equivocal Equivocal samples should be repeated
Samples that remain equivocal after repeat testing should
be repeated by an alternative method or another sample
should be collected
>1.1 Positive Presence of detectable dengue NS1 antigen
Dengue serology assays should be performed on follow-up
samples to confirm dengue infection
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 IMMUNOLOGICAL TESTS
IgM-capture enzyme-linked immunosorbent assay (MAC-ELISA)

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 IMMUNOLOGICAL TESTS
IgG ELISA

This assay uses the same antigens as the MAC-ELISA

Used for the detection of recent or past dengue
infections (if paired sera are collected within the
correct time frame)
Cannot be used to identify the infecting dengue
serotype

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 IMMUNOLOGICAL TESTS
IgM/IgG ratio

Used to distinguish primary infection from secondary

dengue infection

Primary >1.2
Secondary <1.2

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 IMMUNOLOGICAL TESTS
Haemagglutination-inhibition (HI) test
Based on the ability of dengue antigens to agglutinate red
blood cells (RBC) of ganders or trypsinized human O RBC
Anti-dengue antibodies
in sera can inhibit
agglutination and the
potency of this inhibition
is measured in an HI test

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 IMMUNOLOGICAL TESTS
Haemagglutination-inhibition (HI) test
The assay does not discriminate between infections by
closely related flaviviruses nor between immunoglobulin
isotypes
The response to a primary infection is characterized by
the low level of antibodies in the acute-phase serum
drawn before day 5 and a slow elevation of HI antibody
titres thereafter
During secondary dengue infections HI antibody titres
rise rapidly, usually exceeding 1:1280

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 IMMUNOLOGICAL TESTS
Complement fixation (CF)
test
Based on the principle
that the complement is
consumed during antigen-
antibody reactions
Not widely used for
routine dengue diagnostic
serology
It is more difficult to
perform and requires
highly trained personnel
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 IMMUNOLOGICAL TESTS
Neutralization test (NT)
The most specific and sensitive serological test for dengue
viruses used for determining the immune protection
The common protocol used in most dengue laboratories is the
serum dilution plaque reduction neutralization test (PRNT)
The major disadvantages of this technique are the expense
and time required to perform the test, and the technical
difficulty involved since it requires cell culture facility
It is of great use in the development of vaccines and their
efficacy trials

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 IMMUNOLOGICAL TESTS
Neutralization test (NT)

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 HEMATOLOGICAL TESTS
Standard haematological parameters such as platelet
count and haematocrit are important and are part of
the biological diagnosis of dengue infection
should be closely monitored

Thrombocytopenia may be occasionally observed in

DF but is a constant feature in DHF; usually found
between the 3rd and 8th day of illness often before
or simultaneously with changes in Hct

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 HEMATOLOGICAL TESTS
Haemoconcentration with an increase in the
haematocrit 20% (for the same patient or for a
patient of the same age and sex) is considered to be
a definitive evidence of increased vascular
permeability and plasma leakage

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THANK YOU
 PATHOGENESIS
Antibody Dependent Enhancement

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 PATHOGENESIS
Antibody Dependent Enhancement

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 IMMUNOLOGICAL TESTS
Panbio Dengue Duo Casette

Principle
When present in the patient sample, dengue-specific IgM or IgG
antibodies bind to anti-human IgM or IgG antibodies immobilized
in two lines across the cassette
Membrane colloidal gold complexes containing recombinant
dengue 1-4 antigens are captured by the bound patients IgM or
IgG to give visible pink line(s)
A procedural control is included to indicate that the assay has been
performed correctly

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 IMMUNOLOGICAL TESTS
Panbio Dengue Duo Casette

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 IMMUNOLOGICAL TESTS
SD Bioline Dengue Duo

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 IMMUNOLOGICAL TESTS
SD Bioline Dengue Duo

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