Diagnostic de la dengue

1

Outline

• • • • • • • 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 kinetics 1

Primary infection Secondary infection Symptoms Symptoms

IgG IgM NS1 NS1 Viremia

Sting

Viremia

Sting

IgM ?

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.

0 J5 J7-10 IgG response is higher Low levels of detectable IgM

IgG

J15-21 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 Options 2

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

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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

• Inoculation of either mosquitoes (e.g.

T. splendens) ,

cell cultures, namely C6/36, a clone of

Ae.Albopictus

cells.

• Inoculation of mammalian cultures, namely vero cells, LLCMK2 and BHK21.

insect Time: 7 days

Confirmation of dengue infection

• 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.

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 ELISA

4 Enzyme Linked Immunosorbent Assay is based on immunologic reaction antigen-antibody.

sample

E

anti-NS1 Antibody

NS1

ELISA Sandwich

• Characteristics vary by kit •Sensitivity: 58,1 to 93,3% •Specificity: 97,9 to 100% • Time: 2 hours • Not serotype-specific Anti-NS1 Ab conjugated with enzyme

S E Coloration E S E S

= enyme = substrate

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 Monoclonal anti-NS1 antibodies ● The serum migrates by capillarity in a nitrocellulose membrane. ● When the antigen is present, it binds the specific antibodies attached to the membrane and a colored line appears (

T

).

● 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.

15 mn 50 µL serum or plasma

Positive result Negative result Invalid

T C T C T C

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)

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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.

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Direct diagnosis Genome detection

3 Extraction of RNA

RT-PCR

RNA Reverse transcriptase cDNA 94 °C

Denaturation

+ probes

Probes hybridation

+ dNTP + Taq polymerase A cycle

Elongation 2 copies

● PCR amplificates a tiny quantity of viral genom presents in the plasma, making it detectable.

● The PCR products are identified by electrophoresis or using specific probes.

Sensibility: 80 to 100% Specificity: 100% Time: 4 hours N cycles Detection, identification and typing 2 N copies 3. WHO Regional Office for South-East Asia. Comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever. 2011.

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Serological tools

• ELISA • Rapid test • PRNT • HAI

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Serological diagnosis IgM and IgG detection

●

Indirect ELISA

6 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.

S

Coloration Anti-IgM antibody with enzyme Patient’s IgM DEN antigen

S

Coloration

E

Anti-IgG antibody with enzyme Patient’s IgG 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 13

Serological diagnosis IgM and IgG detection

●

Rapid test

7 ● 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 Incubate 1 hour 2) Add with Vero cells culture in the wells Incubate 4 to 7 days Neutralizing antibodies Virus neutralized Absence of neutralizing antibodies Virus not neutralized Cellular death 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)

● 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.

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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).

It is sensitive and easy to perform.

HI antibodies persist up to 50 years.

This test is mainly used for sero-epidemiologic studies.

Haemagglutination Inhibition of heamagglutination Antigens + RBC + + antibody 1. WHO. Dengue. Guidelines for diagnosis, treatment, prevention and control. New edition 2009.

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Interpretation of dengue diagnostic test

10

Highly suggestive

One of the following: • IgM+ in a single serum sample.

• IgG+ in a single serum sample with a HI titre of 1280 or greater.

Confirmed

One of the following: • RT-PCR+ • 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 limitations

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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

Viral isolation and identification RNA detection NS1 Antigen detection

Advantages

• Confirmed infection • Specific • Identifies serotypes • Confirmed infection • Early • Sensitive and specific • Identifies serotype and genotype • Results in 24–48 hours • Confirmed infection • Early appearance • Easy to perform • Less expensive than virus isolation or RNA detection

Limitations

• 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 • Requires acute sample (0–5 days post onset) • Requires expertise and expensive laboratory equipment • Does not differentiate between primary and secondary infection • May not be as sensitive as virus isolation or RNA detection IgM or IgG seroconversion • Confirmed infection • Least expensive • Easy to perform • 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 detection (single sample) • Identifies probable dengue cases • Useful for surveillance, tracking outbreaks and monitoring effectiveness of interventions • 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. 22

Diagnosis Algorythm

3

< Day 5 Virus isolation NS1 detection RT-PCR Suspect case ≤ Day 5 NS1 detection RT-PCR

±

IgM > Day 5 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; 23