Interference and Interpretation of Immunoassays in DHF
Interference and Interpretation of Immunoassays in DHF Francisca Srioetami Tanoerahardjo
Seminar Sehari IDI Cabang Bekasi
Outline
Dengue Virus
Diagnostic Test for Dengue Infection
Interpretation of Diagnostic Test
Interference of Immunoassays
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 has been developed to support patient management and disease control
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 membranes, called replication complex (RC). These RCs seem to contain viral proteins, viral RNA and host cell factors.
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
Future Development
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
Expensive and time consuming (2 weeks to complete 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.
Dash PK, Parida MM, Saxena P, et al. Reemergence of dengue virustype-3 subtype-III) in India: Implication for increase incidence of DHF & DSS. Virology Journal 2006:3:55 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 Yong YK, Thayan R, Chong HT, et al. Rapid detection and serotyping of dengue virus by multiplex RT-PCR and real-time SYBR green RT-PCR. 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.
Singapore Med J 2007;48:662-8.
Time-line correlation:
primary & secondary dengue and diagnostic test Detection of Antigens NS1 detection by ELISA
● Enzyme Linked Immunosorbent Assay is based on immunologic reaction
● antigen-antibody.
- Characteristics vary by kit
ELISA Sandwich anti-NS1
- Sensitivity: 58,1 to 93,3%
sample Antibody
- Specificity: 97,9 to 100%
- Time: 2 hours
- Not serotype-specific
NS1 E Anti-NS1 Ab conjugated with enzyme
Coloration E E E = enyme
S = substrate
S S E S = coloration dengue.2009 professionnels.Détection de l'antigène NS1 de la 4. HAS. Service évaluation des actes + .
● NS1 detection by Immunochromatography
Detection of Antigens
● Combination with detection of IgG/IgM.
- Characteristics vary by kit
- Sensitivity: 58,1 to 93,3%
- Specificity: 97,9 to
- Time: 2 hours
- Not serotype-specific
4. HAS. Service évaluation des actes professionnels.Détection de l'antigène NS1 de la dengue.2009 . NS1 Ag IgM/IgG Reading result at 15-20 minutes
Serological test
ELISA
Rapid test
PRNT
HAI 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 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.6. Guzman MG, Kouri G. Dengue diagnosis, advances and challenges. Int J Infect Dis 2004;8(2):69-80 IgM and IgG detection ● Sensibility and specificity of assays are strongly influenced by the quality of the antigen used and can
7 vary greatly between commercially available products.
● 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 interpreted7 cautiously. Microbiol 2010;8(12 Suppl):S30-S38
7. Peeling RW. et al. Evaluation of diagnostic tests: dengue.Nat Rev
IgM and IgG detection
7 ● Rapid test
● 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. Rev Microbiol 2010;8(12 Suppl):S30-S38 Peeling RW. et al. Evaluation of diagnostic tests: dengue.Nat Serological diagnosis
9 Plaque Reduction Neutralization Test (PRNT) ● 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 human antibodies to dengue viruses. 2007. WHO. Guidelines for plaque reduction neutralization testing for Serological diagnosis
Plaque Reduction Neutralization Test (PRNT)
9 2) 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.
Absence of neutralizing antibodies 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 9 compared to viral load in wells incubated without antibody.
Serial dilutions
1/2 1/4 1/8
Wells Plaque of cellular lyse
reduction
human antibodies to dengue viruses. 2007.9. WHO. Guidelines for plaque reduction neutralization testing for
1 Haemagglutination Inhibition test (HAI) ● 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 control. New edition 2009. WHO. Dengue. Guidelines for diagnosis, treatment, prevention and accessibility and confidence
Hematological 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.
Jyothi P, Metri BC. Correlation of serological markers and platelet count in the diagnosis of Dengue virus infection. Adv Biomed Res 2015;4:26 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
4. IgG seroconversion in paired sera or fourfold IgG titer increase in paired sera
Interpretation of Dengue Diagnostic Test
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.
Interpretation of Dengue Diagnostic Test
Interference of Immunoassays
A relatively rare but still important problem
Interference that alter the measurable analyte concentration in sample
Interference that alter antibody binding
Interference due to other disease
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
Possible pathogenic effect of NS1 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 namun perlu diwaspadai terutama bila mempengaruhi hasil pemeriksaan yang berakibat pada kurang tepatnya penanganan kasus