Dengue Rapid Test for IgG and IgM Detection: Introduction, Principle, Test Requirements, Procedure, Result Interpretation, and Limitations

Introduction of Dengue IgG and IgM Rapid Test

Dengue rapid test (RDT) uses for the detection of immunoglobulin G(IgG) and immunoglobulin M (IgM) where IgM is responsible for the past while IgG is for present infection as shown above picture. Dengue is a mosquito-borne viral disease and the dengue virus is transmitted by female mosquitoes mainly Aedes aegypti and, to a lesser extent, Aedes albopictus. This infection is widespread throughout the tropics, with local variations in risk influenced by rainfall, temperature, relative humidity, and unplanned rapid urbanization. Severe dengue was first recognized in the 1950s during dengue epidemics in the Philippines and Thailand. However, about half of the world’s population is now at risk. There are an estimated 100-400 million infections each year.

The causative agent of dengue is a virus of the Flaviviridae family and there are four distinct serotypes of the virus that cause dengue DENV-1, DENV-2, DENV-3, and DENV-4. Recovery from infection is believed to provide lifelong immunity against that serotype. However, cross-immunity to the other serotypes after recovery is only partial and temporary. Subsequent infections called secondary infections by other serotypes increase the risk of developing severe dengue. There is no specific treatment for dengue/severe dengue.  Early detection of disease progression associated with severe dengue, and access to proper medical care lowers fatality rates of severe dengue to below 1%.

Signs and symptoms of dengue-Dengue should be suspected when a high fever (104°F) is accompanied by 2 of the following symptoms during the febrile phase: severe headache, pain behind the eyes muscle, and joint pains, nausea, vomiting, swollen glands, and rash. Whereas in severe dengue- A patient enters what is called the critical phase normally about 3-7 days after illness onset. It is at this time, when the fever is dropping (<100°F) in the patient, that warning signs associated with severe dengue can manifest. It is a potentially fatal complication, due to plasma leaking, fluid accumulation, respiratory distress, severe bleeding, or organ impairment. Warning signs of severe dengue that doctors should look for are severe abdominal pain, persistent vomiting, rapid breathing, bleeding gums, fatigue, restlessness, and blood in vomit.

The Dengue IgG/IgM Rapid Test is a solid-phase immunochromatographic assay for the rapid, qualitative, and differential detection of IgG and IgM antibodies to dengue virus in human serum, plasma, or whole blood. This test is intended for professional use as an aid in the presumptive diagnosis of primary and secondary dengue infection. The immune response includes IgM antibodies produced on the 3rd to 5th day of symptoms and persist for 30 to 60 days. IgGs appear on the 14th day and persist for life. Secondary infections often result in high fever and in many cases hemorrhagic events and circulatory failure. Secondary infections show that IgGs rise within 1 to 2 days after the onset of symptoms and induce IgM response after 20 days of infection.

Principle of Dengue Rapid Test

The Dengue IgG/IgM Rapid Test is designed to simultaneously detect and differentiate IgG and IgM antibodies to the dengue virus. This test also can detect all 4 dengue serotypes by using a mixture of recombinant dengue envelope proteins. The Dengue IgG/IgM test device has 3 pre-coated lines, “G” (Dengue IgG Test Line), “M” (Dengue IgM Test Line), and “C” (Control Line) on the surface of the membrane. All three lines in the result window are not visible before applying any samples. The “Control Line” is used for procedural control. The Control line should always appear if the test procedure is performed properly and the test reagents of the control line are working. Purple “G” and “M” lines will be visible in the result window if there are enough IgG and/or IgM antibodies to the dengue virus in the sample.

If IgG and/or IgM antibodies to the dengue virus are not present in the sample, there is no color appearance in “G” and/or “M”. When a specimen is added to the sample well, anti-Dengue IgGs and IgMs in the specimen will react with recombinant dengue virus envelope proteins-colloidal gold conjugates and forms a complex of antigen-antibodies. As this complex migrates along the length of the test device by capillary action, it will be captured by the relevant anti-human IgG and or anti-human IgM immobilized in two test lines across the test device and generate a colored line.

Test Requirements for Dengue Rapid Test

1. Test kit contains

• Dengue IgG/IgM test device individually foil pouched with a desiccant
• 1 test strip includes –

1. Gold conjugates: Recombinant dengue virus envelope protein-gold colloid
2. Test Line “G”: Mouse monoclonal anti-human IgG,
3. Test Line “M”: Mouse monoclonal anti-human IgM
4. Control Line: Rabbit anti-dengue IgG

• Assay diluent ( 100 mM Phosphate buffer 5 mL with preservative, Sodium azide 0.01 % w/w)
• Capillary pipette 10 µL
• Package insert

2. Additional needed materials, not provided are-

• Clock
• Specimen (serum/plasma)
• Lancet and alcohol pad for disinfection ( optional for spot sample collection)
• Waste bin

Dengue Rapid Test Procedure

1. Allow all kit components and specimens to room temperature ( if anything is stored in the refrigerator or freezer)  prior to testing.
2. Remove the test device from the foil pouch; place it on a flat, dry surface.
3. Add 10 µL of serum, plasma, or whole blood specimen into the square sample well-marked “S”. with the help of a capillary pipette.
4. Put 3 to 4 drops (about 90 – 120 µL) of assay diluent into the assay diluent well-round shaped. as shown above picture.
5. Interpret test results in 15 to 20 minutes.

Result and Interpretation of Dengue Rapid Test

Result	Interpretation
Dengue IgM positive	The control line (C) and IgM line (M) are visible on the test device. This is positive for IgM antibodies to the Dengue virus. This is indicative of primary dengue infection.
Dengue IgG positive	The control line (C) and IgG line (G) are visible on the test device. This is positive for IgG antibodies. This is indicative of secondary or previous dengue infection.
Dengue IgG and IgM Positive	The control line (C), IgM (M), and IgG line (G) are visible on the test device. This is positive for both IgM and IgG antibodies. This is indicative of a late primary or early secondary dengue infection.
Test Negative	The control line is only visible on the test device. No IgG and IgM antibodies were detected. Retest in 3-5 days if dengue infection is suspected.
Test Invalid	The control line fails to appear. Insufficient specimen volume or incorrect procedural techniques are the likeliest reasons for control line failure. Repeat the test using a new test device.

Limitations of the Dengue Rapid Test

1. This test detects the presence of antibodies to the dengue virus in the specimen and should not be used as the sole criterion for the diagnosis of dengue virus infection.
2. In early infections and some secondary infections, detectable levels of IgM antibodies may be below. Some patients may not produce detectable levels of antibodies within the first seven to ten days after infection. If clinical symptoms persist, patients should be re-tested in 3-4 days with the first specimen.
3. Serological cross-reactivity across the flavivirus group (Dengue virus, St. Louis encephalitis, Japanese encephalitis, West Nile, and yellow fever virus) is common.
4. As with all diagnostic tests, all results must be considered with other clinical information available to the physician.
5. If the test result is negative and clinical symptoms persist, additional follow-up testing using other clinical methods is recommended. A negative result does not preclude the possibility of early infection with the dengue virus.
6. The test procedure, precautions, and interpretation of results for this test must be followed strictly when testing. The “Control Line” is used for procedural control. The Control line should always appear if the test procedure is performed properly and the test reagents of the control line are working. It confirms sufficient specimen volume and correct procedural technique. A clear background is also required.

Keynotes on Dengue Rapid Test

1. Aedes aegypti and Aedes albopictus are also vectors of chikungunya, yellow fever, and Zika viruses.
2. NS1 ( non-structural protein) antigenemia is detectable within 24 hours of infection and up to 9 days following symptom onset. The dengue virus NS1 antigen can be detected by ordering  Dengue Virus NS1 Antigen, Serum.
3. The sensitivity and specificity rate of test kits vary from manufacturer to manufacturer. e.g. DRG® Dengue IgG/IgM Rapid Test kit Sensitivity is 91.2% and specificity of 90% whereas InBios DV IgM EIA Sensitivity: is 87.5% and Specificity: is 100% and  InBios DV IgG EIA Sensitivity: is 94.4% and Specificity: 100%.

Dengue Rapid Test for IgM, IgG, and NS1 antigen Detection Test Device

Dengue Rapid Test for IgM, and IgG Detection

Dengue Rapid Test for IgM, IgG, and NS1 antigen Detection

Dengue Rapid Test for IgM, IgG, and NS1 antigen Detection

Dengue NS1 antigen-Positive

Bibliography of Dengue Rapid Test

1. https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue
2. https://www.sceti.co.jp/images/psearch/pdf/DRG_RAP4894_p.pdf
3. https://www.ncbi.nlm.nih.gov/books/NBK143156/
4. http://www.rapidtest.com/index.php?i=Dengue-Rapid-Test-&id=446&cat=26
5. https://www.mayocliniclabs.com/testcatalog/Clinical+and+Interpretive/83865
6. https://www.who.int/csr/resources/publications/dengue/034-47.pdf?ua=1
7. https://www.cdc.gov/dengue/healthcare-providers/testing/antigen-detection.html