Diagnostic
de la dengue
1
Outline
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Biological markers kinetics
Direct diagnosis
• Viral isolation
• Antigen detection
• RNA detection
Indirect diagnosis
• ELISA
• Rapid test
• PRNT
• HAI
Dengue confirmation
Distinction primary/secondary infection
Advantages and limitations
Algorythm
2
Biological markers kinetics1
Primary infection
Symptoms
Secondary infection
IgM
NS1
Viremia
Symptoms
IgG
NS1
Viremia
Sting
Sting
0
J5 J7-10
J30
3-6 mois
IgM are produced earlier than IgG. A four
fold or greater increase in IgG antibody
levels demonstrates a seroconversion.
IgG
IgM ?
0
J5
J7-10
J15-21
IgG response is higher
Low levels of detectable IgM
Virus and viral antigens are detectable in blood during the first days of illness.
1. WHO. Dengue. Guidelines for diagnosis, treatment,
prevention and control. New edition 2009
3
Laboratory Diagnostic Options2
NS=Dengue NS1(non-structural protein 1) antigen; RT-PCR=Reverse transcription polymerase chain reaction;
IgM=immunoglobulin M; IgG=Immunoglobulin G; ELISA=enzyme-linked immunosorbent assay
2. Simmons CP, et al. N Engl J Med 2012;366:1423–32
4
Direct diagnosis
• Viral isolation
• Antigen detection
• RNA detection
5
Direct diagnosis
Virus isolation
● Direct inoculation of mosquito, mosquito cell culture and mammalian cultures
are used to detect the virus from serum or plasma.1
● Dengue virus serotype is identified by immunofluorescence or molecular
testing.3
Recommanded methods
Confirmation of dengue infection
,
• Inoculation of either mosquitoes (e.g. T. splendens) insect
cell cultures,
namely C6/36, a clone of Ae.Albopictus
cells.
• Inoculation of mammalian cultures,
namely vero cells, LLCMK2 and
BHK21.
• Presence of antigens in cells
demonstrated by
immunofluorescence (IFA). Viral
titre is confirmed by RT-PCR or plaque
assay.
• Cytopathic effect and plaque
formation in mammalian
cells.
Time: 7 days
3. WHO Regional Office for South-East Asia. Comprehensive
guidelines for prevention and control of dengue and dengue
haemorrhagic fever. 2011.
6
Direct diagnosis
NS1 detection by ELISA4
● Enzyme Linked Immunosorbent Assay is based on immunologic reaction
antigen-antibody.
sample
anti-NS1
Antibody
ELISA
Sandwich
• Characteristics vary by kit
•Sensitivity: 58,1 to 93,3%
•Specificity: 97,9 to 100%
• Time: 2 hours
E
Anti-NS1 Ab
conjugated
with enzyme
• Not serotype-specific
NS1
E
E
Coloration
S
E = enyme
S = substrate
S
E
+
S = coloration
4. HAS. Service évaluation des actes
professionnels.Détection de l'antigène NS1 de la
dengue.2009.
7
Direct diagnosis
NS1 detection by Immunochromatographic Test (ICT)
T C
Strip test
migration
Deposit of serum
● The serum migrates by capillarity in a
nitrocellulose membrane.
Monoclonal anti-NS1
antibodies
15 mn
Positive result T C
● When the antigen is present, it binds the
specific antibodies attached to the
membrane and a colored line appears (T).
Negative result T C
● The appearence of the control line (C)
indicates that the migration was correctly
made. If it is not present, the test is
considered invalid and must be repeated.
Invalid
T C
50 µL serum
or plasma
5. Lima MR et al. A New Approach to Dengue Fatal Cases
Diagnosis: NS1 Antigen Capture in Tissues. PLoS Negl Trop
Dis 2011;5(5):e1147
8
Direct diagnosis
NS1 detection by Immunochromatographic Test (ICT)4
Cassette test
The principle is the same; drops of serum are
deposited in the sample well.
ICT
• Sensitivity: 58,4 to 80%
• Specificity: 100%
Immunochromatography is less
time consuming and easier to
perform than ELISA
4. HAS. Service évaluation des actes professionnels. Détection de
l'antigène NS1 de la dengue. 2009.
9
Direct diagnosis
Genome detection3
● PCR amplificates a tiny quantity of
viral genom presents in the plasma,
making it detectable.
Extraction of RNA
RT-PCR
RNA
Reverse transcriptase
94°C
● The PCR products are identified by
electrophoresis or using specific
probes.
+ probes
Sensibility: 80 to 100%
cDNA
Denaturation
Probes
hybridation
Specificity: 100%
+ dNTP
+ Taq polymerase
A cycle
Time: 4 hours
Elongation
N cycles
2 copies
Detection, identification and
typing
2N copies
3. WHO Regional Office for South-East Asia. Comprehensive
guidelines for prevention and control of dengue and dengue
haemorrhagic fever. 2011.
10
Serological tools
• ELISA
• Rapid test
• PRNT
• HAI
11
Serological diagnosis
IgM and IgG detection
● Indirect ELISA6
Microwells are coated with purified dengue virus antigen type 1-4. Anti-dengue
antibodies of sera bind to the viral antigens. Anti-IgM or Anti-IgG antibodies
conjugated with enzyme are added to reveal the binding.
Coloration
S
Coloration
Anti-IgM
antibody
with enzyme
S
E
Anti-IgG
antibody
with enzyme
Patient’s IgM
Patient’s IgG
DEN antigen
DEN antigen
6. Guzman MG, Kouri G. Dengue diagnosis, advances and
challenges. Int J Infect Dis 2004;8(2):69-80
12
Serological diagnosis
IgM and IgG detection
● Sensibility and specificity of assays are strongly
influenced by the quality of the antigen used and can
vary greatly between commercially available products.7
● Because of an importantly cross-reactivity, these tests
cannot be used to identify the infecting dengue virus
serotypes. IgG Antibodies also cross react between
dengue and other flaviviruses, therefore the result must
be interpreted cautiously.7
7. Peeling RW. et al. Evaluation of diagnostic tests: dengue.Nat Rev
Microbiol 2010;8(12 Suppl):S30-S38
8. Falconar AK and al. Clin Vaccine Immunol 2006;13: 1044-51
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Serological diagnosis
IgM and IgG detection
● Rapid test7
● ICT (15 to 90 mn)
Sensitivity: 21% to 99%
Specificity: 77% to 98%
The ELISA tests show greater sensitivity in detecting dengue-specific
antibodies than the rapid tests, but the rapid tests are field friendly, with the
results available in a shorter timeframe.
7. Peeling RW. et al. Evaluation of diagnostic tests: dengue.Nat
Rev Microbiol 2010;8(12 Suppl):S30-S38
14
Serological diagnosis
Plaque Reduction Neutralization Test (PRNT)9
● PRNT is the simplest and most widely used way to detect and measure
neutralizing antibodies specific of each of four serotypes.
● PRNT or other neutralization assays (such as micro-neutralization) are
the most serotype-specific and sensitive serological tests. But they have
some limitations for diagnosis especially in secondary and subsequent
infections: an increase of titers of antibodies against prior infection serotypes
is often observed (antigenic sin)
● It is more widely used in sero-epidemiological cohort studies examining
non-incidental dengue infection using annual blood draws
9. WHO. Guidelines for plaque reduction neutralization testing
for human antibodies to dengue viruses. 2007.
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Serological diagnosis
Plaque Reduction Neutralization Test (PRNT)9
● Neutralizing antibodies are able to inactivate the virus and to prevent permissive
cells infection and death.
● They appear 2 to 3 weeks after the onset of symptoms and are detectable
for a long time.
● The serum specimen being tested is subjected to serial dilutions prior to
mixing with a standardized amount of virus.
1)
Add DENV virus with each serial
dilution tube
Neutralizing
antibodies
Incubate 1 hour
Absence of
neutralizing
antibodies
2) Add with Vero cells culture
in the wells
Incubate 4 to 7 days
Virus neutralized
Virus not neutralized
Cellular death
9. WHO. Guidelines for plaque reduction neutralization testing
for human antibodies to dengue viruses. 2007.
16
Serological diagnosis
Plaque Reduction Neutralization Test (PRNT)
● Test measures the antibodies titer by linear regression analysis or
determines the highest dilution that results in 50% reduction of plaque count
compared to viral load in wells incubated without antibody.9
Serial dilutions
1/2
1/4
1/8
Wells
Plaque of
cellular lyse
reduction
9. WHO. Guidelines for plaque reduction neutralization testing
for human antibodies to dengue viruses. 2007.
17
Serological diagnosis
Haemagglutination Inhibition test (HAI)1
● HAI test is based on the ability of dengue antigens to agglutinate red blood
cells (RBC). It measures inhibition of this agglutination caused by anti-dengue
antibodies (IgG or IgM).
Inhibition of
● It is sensitive and easy to perform.
Haemagglutination
heamagglutination
HI antibodies persist up to 50 years.
This test is mainly used for
sero-epidemiologic studies.
+
Antigens
RBC
+ +
antibody
1. WHO. Dengue. Guidelines for diagnosis, treatment, prevention
and control. New edition 2009.
18
Interpretation of dengue diagnostic test10
Highly suggestive
Confirmed
One of the following:
One of the following:
• IgM+ in a single serum sample.
• RT-PCR+
• IgG+ in a single serum
sample with a HI titre of 1280
or greater.
• Virus culture+
• NS1+
• IgM seroconversion in paired sera.
• IgG seroconversion in paired sera
or four-fold IgG titre increase in
paired sera.
10. Jaenisch T., Wills B. Results from the DENCO study.
TDR/WHO Expert Meeting on Dengue Classification
and Case Management. WHO, Geneva, 2008.
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How to distinguish primary and secondary infections
● IgM/IgG ratio is used to differentiate a primary from a secondary infection.8
• Hight: >2,6
Primary dengue infection
• Low: < 2,6
Secondary dengue infection
● PRNT is the only serological test that can provide serotype- specific results. It
may also provide information on history of infection (primary, secondary and
further) though interpretation of secondary or further infections can be
challenging, due to cross-reactivity by the presence of heterotypic antibodies.
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Advantages and limitations7
7. Peeling RW. et al. Evaluation of diagnostic tests: dengue.Nat
Rev Microbiol 2010;8(12 Suppl):S30-S38
21
Advantages and limitations of different dengue diagnosic tests(adapted from 7)
Diagnostic tests
Advantages
Limitations
Viral isolation and identification
• Confirmed infection
• Specific
• Identifies serotypes
• Requires acute sample (0–5 days post onset)
• Requires expertise and appropriate facilities
• Takes more than 1 week
• Does not differentiate between primary and secondary
infection
• Expensive
RNA detection
•
•
•
•
•
• Requires acute sample (0–5 days post onset)
• Requires expertise and expensive laboratory equipment
• Does not differentiate between primary and secondary
infection
NS1 Antigen detection
• Confirmed infection
• Early appearance
• Easy to perform
• Less expensive than virus isolation
or RNA detection
Confirmed infection
Early
Sensitive and specific
Identifies serotype and genotype
Results in 24–48 hours
IgM or IgG seroconversion
• Confirmed infection
• Least expensive
• Easy to perform
IgM detection (single sample)
• Identifies probable dengue cases
• Useful for surveillance, tracking
outbreaks and monitoring
effectiveness of interventions
• May not be as sensitive as virus isolation or RNA
detection
• IgM levels can be low in secondary infections
• Confirmation requires two or more serum samples
• Can differentiate between primary and secondary
infection*
• IgG specificity is lower due to cross reactions among
flaviviruses*
• IgM levels can be low in secondary infections
*Primary
infection: IgM-positive and IgG-negative (if samples are taken before day 8–10); secondary infection: IgG should be higher
than 1,280 using haemagglutination inhibition in convalescent serum.
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Diagnosis
Algorythm3
Surveillance
Diagnostic
Suspect case
< Day 5
≤ Day 5
> Day 5
Virus isolation
NS1 detection
RT-PCR
NS1 detection
RT-PCR
± IgM
IgM
Serotype
identification
Serotype
identification
Sequencing and Epidemiology
NS1=dengue NS1 antigen; RT-PCR=reverse transcription polymerase chain reaction;
IgM=immunoglobulin M
3. World Health Organization. Dengue guidelines for diagnosis, treatment, prevention and
control;
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