Comparison of Laboratory - Based Techniques for the Detextion of Dengue Infections

Pimentel,Buerano, Inoue, Matias, Natividad

 Vector disease Aedes aegypti Mild dengue fever (DF) or Dengue

hemorrhagic fever (DHF)

Dengue

can lead to Dengue shock syndrome

(DSS)

RNA virus Flaviviridae

Mechanism
Mosquito bite to salivary glands White blood cells Signal Proteins (interferon) in severe infection, organs are affected fluid leak

Tests
Serological assay PCR Cell culture DIAGNOSIS

Methodology

 112 patients with thrombocytopenia

from San Lazaro Hospital

Sampling

Platelet count Normal - 150,000/mm (not included) Slight decrease - <100,000/mm to

150,000/mm

Intermediate decrease - >50,000 to

100,000/mm

Severe Decrease - < 50,000/mm

Sampling

3 to 5 ml blood sample within 15 days
Centrifugated at 1,800 rpm for 10 minutes at 4 degrees Celsius Collect Plasma and Buffy Coat and disacard RBC Wash with Phosphate Buffered Saline (PBS) at 1,300 rpm , resusped in 50 to 100 ul PBS Osmotic Equilibrium Separate components RBC does not contain virus

Immunofluorescence Antiobody Test (IFAT)

Fix in cold acetone and airdried
20 ul of Blockace solution Slides placed at 37 degrees Celsius and washed with PBS Addition of 20 ul of mouse monoclonal anti-flavivirus antibodies 20 ul of FITC - labeled anti mouse IgG

blocking reagent to prevent non - specific binding Optimum Temperature

Detection, binds to target Dye, binds to antibody

Result
A: plasma specimen (test sample) B: DEN2 virus (positive control) C: Mock-infected cells (blank) Fluorescence (neon green color) indicates presence of dengue virus.

Result

Results
100% detection in first two days declined to 0% Time dependent

Infectivity Assay

prepared BHK - 21 cells, spread inoculum
Addition of 50 ul of human IgG anti - dengue antibody, washing and incubation Addition of 50 ul horse radish peroxidase conjugated anti human IgG Addition of 50 ul 0.05% Diaminobenzidine and Hydrogen peroxide cell to be inoculated with infected culture fluid

primary antiobody

secondary antibody with peroxidase, binds DAB and oxidize it, producing brown color as stain

Results

IgM-capture ELISA

Each well of a 96-well plate was coated with 100 uL of goat antihuman IgM in 0.05 M carbonate-bicarbonate buffer, pH 9.6, with 0.01% NaN3, incubate.

Preparation of wells

Wash plate with PBSTween with interval of three minutes

To remove excess

IgM-capture ELISA

Plasma from dengue patients, (+) control, (-) control were put in designated well, incubated for one hour at 37 degrees Celsius

Addition of test specimens to the well

Wash wells with PBS Tween

To eliminate non-specific bindings

IgM-capture ELISA

100 uL of tetravalent dengue viral antigen was added to each plate, incubate, wash

To form complex

Each plate was reacted with diluted horseradish peroxidase

To hydrolyze the complex formed

IgM-capture ELISA

Add phenylenediamine dihydrochloride (OPD), 0.03% H2O2 in 0.05M citrate phosphate buffer
Stop reaction, read plate at 492 nm

For color reaction

Colour development is indicative of the presence of anti-dengue IgM antibodies in the test sample.

Results

Reverse Transcription-PCR

RNA was isolated, air dried, dissolved in diethylpyrocarbonatetreated water (DEPC), incubated Heated RNA was added to 10 uL reverse propagation mix, incubated for 1 hr PCR

To linearize RNA

For cDNA synthesis

Reverse Transcription-PCR

Agarose Gel Electrophoresis of PCR products

To detect genome similarities as compared to the standards

Results

Results

Antigen Sandwich ELISA

Each well was coated with anti-flavivirus IgG, incubated overnight, washed Test specimens were put Put HRPO conjugated anti-flavivirus IgG

Preparation of well

To complex antigen and virus React HRPO and antigen, so that virus is sandwiched between antigens Detect presences of dengue virus

View under 492 nm

Result