Specimen Collection and Preparation: The Dengue Combo Rapid Test Cassette (Whole Blood/Serum/Plasma) can be performed using whole blood, serum, or plasma.
To collect Fingerstick Whole Blood Specimens: Wash the patient's hand with soap and warm water or clean with an alcohol swab. Allow to dry.
Massage the hand without touching the puncture site by rubbing down the hand towards the fingertip of the middle or ring finger.
Puncture the skin with a sterile lancet. Wipe away the first sign of blood.
Gently rub the hand from wrist to palm to finger to form a rounded drop of blood over the puncture site.
Add the Fingerstick Whole Blood specimen to the test cassette by using a 5 μL dropper or micropipette measuring 10 μL for IgG/IgM test and a 25 μL dropper or micropipette measuring 75 μL for NS1 test. The 5 μL or 25 μL droppers provided with the test dispenses approximately 10 μL or 25 μL in one drop even if more blood is aspirated in the dropper.
Separate serum or plasma from blood as soon as possible to avoid hemolysis. Use only clear, non-hemolyzed specimens.
Testing should be performed immediately after specimen collection. Do not leave the specimens at room temperature for prolonged periods. Serum and plasma specimens may be stored at 2-8°C for up to 3 days. For long-term storage, specimens should be kept below -20°C. Whole blood collected by venipuncture should be stored at 2-8°C if the test is to be run within 2 days of collection. Do not freeze whole blood specimens. Whole blood collected by fingerstick should betested immediately.
Bring specimens to room temperature prior to testing. Frozen specimens must be completely thawed and mixed well prior to testing. Specimens should not be frozen and thawed repeatedly.
If specimens are to be shipped, they should be packed in compliance with local regulations for transportation of etiologic agents.
EDTA K2, Heparin sodium, Sodium citrate, and Potassium oxalate can be used as the anticoagulant for collecting the specimen.
Directions for Use: Allow the test, specimen, buffer, and/or controls to reach room temperature (15-30°C) prior to testing.
1. Bring the pouch to room temperature before opening it. Remove the test cassette from the sealed pouch and use it within 1 hour.
2. Place the cassette on a clean and level surface.
For Serum or Plasma specimen: For IgG/IgM: To use a dropper: Hold the dropper vertically, draw the specimen up to the Fill Line approximately 5 μL), and transfer the specimen to the specimen well (S) of the test cassette, then add 3 drops of buffer (approximately 120 μL) to the buffer well (B) and start the timer. Avoid trapping air bubbles in the specimen well.
To use a micropipette: Pipette and dispense 5 μL of serum or plasma to the specimen well (S) of the test cassette, then add 3 drops of buffer (approximately 120 μL) to the buffer well (B) and start the timer.
For NS1: Hold the dropper vertically and transfer 3 drops of serum or plasma (approximately 75 μL) to the specimen well (S), and start the timer.
For Whole Blood (Venipuncture/Fingerstick) specimen: For IgG/IgM: To use a dropper: Hold the dropper vertically, draw the specimen up to about 1cm above the fill line, and transfer 1 drop of whole blood (approximately 10 μL) to the specimen well (S) of the test cassettes, then add 3 drops of buffer (approximately 120 μL) to the buffer well (B) and start the timer.
To use a micropipette: Pipette and dispense 10 μL of whole blood to the specimen well (S) of the test cassette, then add 3 drops of buffer (approximately 120 μL) to the buffer well (B) and start the timer.
For NS1: To use a dropper: Hold the dropper vertically and transfer 3 drops of whole blood (approximately 75 μL) to the specimen well (S), then add 1 drop of buffer (approximately 40 μL) and start the timer.
To use a capillary tube: Fill the capillary tube and transfer approximately 75 μL of fingerstick whole blood specimen to the specimen well (S) of test cassette, then add 1 drop of buffer (approximately 40 μL) and start the timer.
3. Read the results at 10 minutes, do not interpret the results after 20 minutes.
Note: It is suggested not to use the buffer beyond 6 months after opening the vial.
Interpretation of Results: NS1 POSITIVE: *Two colored lines appear. One colored line should be in the control region (C) and another colored line should be in the test region (NS1).
IgG and IgM POSITIVE: *Three colored lines appear. One colored line should be in the control line region (C), and two colored lines should appear in IgG test line region and IgM test line region. The color intensities of the lines do not have to match. The result is positive for IgG and IgM antibodies indicated end stage of primary Dengue infection and early stage of secondary Dengue infection.
IgG POSITIVE: *Two colored lines appear. One colored line should be in the control line region (C), and a colored line appears in IgG test line region. The result is positive for Dengue virus specific-IgG and is probably indicative of secondary Dengue infection.
IgM POSITIVE: *Two colored lines appear. One colored line should be in the control line region (C), and a colored line appears in IgM test line region. The result is positive for Dengue virus specific-lgM antibodies and is indicative of primary Dengue infection.
*NOTE: The intensity of the color in the test line region (NS1 and/or IgG and/or IgM) will vary depending on the concentration of Dengue NS1 antigen and/or IgG and/or IgG present in the specimen. Therefore, any shade of red in the test region should be considered positive.
NEGATIVE: One colored line appears in the control region (C). No line appears inthe test region (IgG/IgM/NS1).
INVALID: Control line fails to appear. Insufficient specimen volume or incorrect procedural techniques are the most likely reasons for control line failure. Review the procedure and repeat the test with a new test cassette. If the problem persists, discontinue using the test kit immediately and contact the local distributor. (See figure.)
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Quality Control: A procedural control is included in the test. A colored line appearing in the control region (C) is the internal procedural control. It confirms sufficient specimen volume and correct procedural technique. Control standards are not supplied with this kit; however, it is recommended that a positive control and a negative control be tested as a good laboratory practice to confirm the test procedure and to verify proper test performance.
Limitations: The Dengue Combo Rapid Test Cassette (Whole Blood/Serum/Plasma) will only indicate the presence of Dengue NS1 antigen and Dengue antibodies in the specimen and should not be used as the sole criteria for the diagnosis of Dengue.
The DIRECTIONS FOR USE and the INTERPRETATION OF RESULTS must be followed closely when testing the presence of dengue antibody/antigen in whole blood, serum, or plasma from individual subjects. Failure to follow the procedure may give inaccurate results.
A negative test result for Dengue NS1 does not preclude the possibility of exposure to or infection with dengue viruses.
A negative result for Dengue NS1 can occur if the quantity of dengue antigen present in the specimen is below the detection limits of the assay, or the dengue antigen that are detected are not present during the stage of disease in which a sample is collected.
Some specimens containing unusually high titer of heterophile antibodies or rheumatoid factor may affect expected results.
If the symptom persists, while the result from Dengue NS1 Rapid Test is negative or non-reactive result, it is recommended to re-sample the patient few days later or test with an alternative test device such as PCR, ELISA.
The results obtained with this test should only be interpreted in conjunction with other diagnostic procedures and clinical findings.
In the early onset of fever, anti-Dengue IgM concentrations may be below detectable levels. For primary infection, an IgM antibody-capture enzyme-linked immune sorbent assay (MACELISA) showed that 80% of the Dengue patients tested exhibited detectable levels of IgM antibody by the fifth day after infection, and 99% of the patients tested IgM positive by day 10. It is recommended that patients be tested within this time. For the secondary infection, a low molar fraction of anti-Dengue IgM and a high molar fraction of IgG that is broadly reactive to flaviviruses characterize the antibodies. The IgM signal may be faint and the cross-reaction in the region of IgG line may appear.
Serological cross-reactivity across the flavivirus group (Dengue 1, 2, 3 & 4, St. Louis encephalitis, West Nile virus, Japanese encephalitis, and yellow fever viruses) is common. Positive results should be confirmed by other means.
The continued presence or absence of antibodies cannot be used to determine the success or failure of therapy.
Results from immunosuppressed patients should be interpreted with caution.
As with all diagnostic tests, all results must be interpreted together with other clinical information available to the physician.
If the test result is negative and clinical symptoms persist, additional testing using other clinical methods is recommended. A negative result does not at any time preclude the possibility of Dengue infection.
The hematocrit of the whole blood should be between 25% and 65%.
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