Interference and Interpretation of

Immunoassays in DHF

Francisca Srioetami Tanoerahardjo

Seminar Sehari IDI Cabang Bekasi

18 April 2015

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 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.

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.

Singapore Med J 2007;48:662-8.

Time-line correlation: primary & secondary dengue and diagnostic test

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

+ = coloration S

S

= enyme

= substrate

E

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 Reading result at 15-20 minutes

Serological test

ELISA

Rapid test

PRNT

HAI

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.

6. Guzman MG, Kouri G. Dengue diagnosis, advances and

challenges. Int J Infect Dis 2004;8(2):69-80

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

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 Rev Microbiol 2010;8(12 Suppl):S30-S38

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

WHO. Guidelines for plaque reduction neutralization testing for

human antibodies to dengue viruses. 2007.

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

compared to viral load in wells incubated without antibody.9

Plaque of cellular lyse

Wells

Serial dilutions

1/4 1/2 1/8

reduction

9. WHO. Guidelines for plaque reduction neutralization testing for

human antibodies to dengue viruses. 2007.

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

antibody

WHO. Dengue. Guidelines for diagnosis, treatment, prevention and

control. New edition 2009.

Comparison of diagnostic tests according to

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

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.

Interpretation of Dengue Diagnostic Test

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