Interference and Interpretation of
Immunoassays in DHF
Francisca Srioetami Tanoerahardjo
Outline
Dengue Virus
Diagnostic Test for Dengue Infection
Interpretation of Diagnostic Test
INTRODUCTION
Primary importance for clinical care of
dengue is
efficient and accurate
diagnosis
early detection of severe cases
case confirmation
differential diagnosis with other infectious
diseases
A range of laboratory diagnostic methods
DENGUE VIRUS
Single-Stranded RNA Viruses Family Flaviviridae
Mosquito borne disease
Four serotypes DENV1-4
Genome : 11000 bases
three structural proteins: C, prM, E;
seven nonstructural proteins: NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5;
short non-coding regions
Replication of DENV induces rearrangements of intracellular
Diagnostic Test for Dengue Infection
Virus Isolation
Cell culture
Nucleic Acid Detection
RT-PCR assays Detection of Antigens
Detection of Antigens
NS1
Serological Test
IgM, IgG
Hematological Test
Thrombosit
Virus Isolation
Definitive test for dengue infection
Lab equipped with tissue culture facilities
Useful only at early phase of illness , blood collected
before day 5 of illness ( before the formation of
Neutralizing antibodies)
During febrile illness
Virus can be isolated from serum, plasma and leucocytes
Post mortem specimens
Virus Isolation
Virus isolation has a poor yield if compared with
molecular test. It is most probably due to the viability
of the virus and the quality of samples.
Nucleic Acid Detection
Methods
Reverse Trancriptase-Polymerase Chain Reaction assays (RT-PCR assays)
Real-time – Reverse Transcriptase-Polymerase Chain Reaction assays (Real-time RT-PCR assays)
Isothermal Amplification Methods
Diagnosis of dengue infection in the early phase (< 5 days
of illness)
Sensitivity 100% in the first 5 days of illness, reduced to
70% by day 6
Determine dengue serotype
Kong YY, Thay CH, Tin TC, et al. Rapid detection, serotyping and quantitation of dengue viruses by TagMan real-time one-step RT-PCR. Journal of Virological Methods 2006;138:123-30.
Time-line correlation:
Detection of Antigens
• Characteristics vary by kit •Sensitivity: 58,1 to 93,3%
•Specificity: 97,9 to 100%
• Time: 2 hours
• Not serotype-specific
●
NS1 detection by ELISA●
Enzyme Linked Immunosorbent Assay is based on immunologic reaction antigen-antibody.E
4. HAS. Service évaluation des actes
professionnels.Détection de l'antigène NS1 de la dengue.2009.
sample anti-NS1 Antibody
E NS1
Coloration S E E S ELISA Sandwich Anti-NS1 Ab conjugated with enzyme
+ S = coloration
S
= enyme = substrate
Detection of Antigens
• Characteristics vary by kit •Sensitivity: 58,1 to 93,3%
•Specificity: 97,9 to 100%
• Time: 2 hours
• Not serotype-specific
●
NS1 detection by Immunochromatography●
Combination with detection of IgG/IgM.4. HAS. Service évaluation des actes
professionnels.Détection de l'antigène NS1 de la dengue.2009.
NS1 Ag IgM/IgG
Serological test
ELISA
Rapid test
PRNT
Serological diagnosis
IgM and IgG detection
E S DEN antigen Patient’s IgG Anti-IgG antibody with enzyme DEN antigen Patient’s IgM Anti-IgM antibody with enzyme S
Coloration Coloration
● 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.
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
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.
Peeling RW. et al. Evaluation of diagnostic tests: dengue.Nat
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
Serological diagnosis
Plaque Reduction Neutralization Test (PRNT)
92) Add with Vero cells culture in the wells
Incubate 4 to 7 days
1) Add DENV virus with each serial dilution tube
Incubate 1 hour
Neutralizing antibodies
Virus neutralized Virus not neutralized Cellular death
● 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.
WHO. Guidelines for plaque reduction neutralization testing for human antibodies to dengue viruses. 2007.
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
Plaque of cellular lyse
Wells Serial dilutions
1/4
1/2 1/8
reduction
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+
RBC Antigens antibodyHematological Test
Thrombocytopenia as a predictive marker
Association of thrombocytopenia in dengue parameter-positive
cases was highly significant when compared to
thrombocytopenia in dengue parameter-negative cases.
Interpretation of Dengue Diagnostic Test
Highly suggestive
One of the following:
1. IgM + in a single serum sample
2. IgG + in a single serum sample with a HI like titre of 1280 or greater
Confirmed
One of the following: 1. RT-PCR +
2. Virus culture +
3. IgM seroconversion in paired sera
NS1-basedcapture test can be applied to distinguish
DENV-1 and DENV-3from other serotype
Dengue NS1 Ag STRIP Kit may be the best kit for
confirming and serotyping dengue infection.
NS1-based tests with diagnostic utility for comfirming dengue infection: a meta-analysis. Zhang H, Li W, Wang J, et al. International Journal of Infectious Diseases 2014;26:57-66.
NS1 as a co factor in virus replication
NS1 engangement with host innate and adaptive immunity
NS1 induction of autoantibodies and a potential role in
pathogenesis
NS1 as a diagnostic biomarker
Muller DA, Young PR. The Flavivirus NS1 protein: Molecular and structural biology, immunology, role in pathogenesis and aplication as a diagnostic biomarker.. Antiviral Research 2013;98:192-208
Amorim JH, Alves RPS, Boscardin SB, et al. The dengue virus non-structural 1 protein: Risk and benefit. Virus Research 2014;181:53-60.
Interference of Immunoassays
A relatively rare but still important problem
Interference that alter the measurable analyte
concentration in sample
Interference that alter antibody binding
Evaluation test for DHF / DSS
Pathogenesis of thrombocytopenia and coagulopathy in
DHF/DSS
Possible pathogenic effect of anti-NS1 cross reactive
antibodies during DENV infection
Summary
Uji diagnostik untuk Dengue masih berkembang dengan
teknologi yang lebih baru
Interpretasi hasil pemeriksaan laboratorium
membutuhkan data klinis dan komunikasi dengan klinisi agar pengelolaan kasus lebih optimal
Interferensi dalam immunoassays walaupun jarang terjadi
