Laboratory Diagnosis of Dengue Viral Infection

This post was most recently updated on August 20th, 2019

Dengue and dengue hemorrhagic fever (DHF) are mosquito-borne viral disease caused by dengue virus, an Arbovirus (Arthropod borne Virus). It is transmitted via the bite of infected female Aedes aegypti  mosquito. Laboratory diagnosis method for confirming dengue viral infection involves use of  one or combination of any of the following four different methods 

1. Microscopy and staining
2. Culture
3. Serology
   1. Detection of antigen
   2. Detection of antibody
4. Molecular diagnosis

Note: These diagnosis methods are used for the diagnosis of any infections (viral, bacterial, parasitic or fungal). The relative importance of particular method differs among infections. Students are expected to know main diagnostic test(s) of the particular infection.

Sample:

1. Early stages of the disease: After the onset of illness, virus can be detected in blood (serum, plasma) or tissues; methods employed are; virus isolation, nucleic acid or antigen detection.
2. At the end of acute phase of infection: Serology is the method of choice.

Note: For virus culture, it is important to keep blood samples cooled or frozen to preserve the viability of the virus during transport from the patient to the laboratory.

1. Microscopy and Staining:  Direct visualization of the virus in the sample (using electron microscopy or via fluorescent staining technique) can be done. This is not the preferred method in diagnostic laboratories.
2. Culture: Virus isolation in cell culture is difficult and is not the commonly used method in diagnostic laboratories because it is a demanding procedure (both in terms of infrastructure and technical expertise).  Virus may be recovered from serum, plasma and peripheral blood mononuclear cells.  Inoculation of a mosquito cell line with patient serum, coupled with nucleic acid assays to identify the recovered virus is commonly used approach in the research lab.

   

3. Serological test: Serological tests are the mainstay in the diagnosis of viral infections. 
   • Detection of viral antigen:
     • Dengue NS1 antigen detection is useful for the diagnosis of acute dengue infections.
     • NS1 antigen has been detected in the serum of DENV infected patients as early as 1-day post onset of symptoms (DPO), and up to 18 DPO.
     • NS1 ELISA based antigen assay is commercially available
     • NS1 assay may also be useful for differential diagnostics between flaviviruses because of the specificity of the assay
4. Detection of  anti-dengue antibodies in serum or other body fluids by ELISA or other rapid tests.  Various methods (IgM/IgG ELISA, Hemagglutination Inhibition Test, or rapid diagnostic kits) are available to detect anti-dengue antibodies;IgM detection:  
   • Useful for the diagnosis of primary dengue infection and in distinguishing dengue from other flavivirus infections.
   • IgM antibodies are detectable in 99% of patients by day 10 after the onset of illness.
   • IgM levels peak about two weeks after the onset of symptoms and then decline to undetectable levels over 2–3 months. 
   • Sensitivity: 65-75% sensitive in single acute serum sample.
     

IgG detection: Tests that detect IgG are useful in diagnosing secondary disease (IgG is the dominant immunoglobulin type in secondary infection). The test is complicated by cross-reactivity of IgG antibodies to heterologous flavivirus antigens (West Nile virus, tick-borne encephalitis virus, yellow fever virus, Zika virus).
Note: To distinguish primary and secondary dengue infections, IgM/IgG antibody ratios are now more commonly used than the haemagglutination-inhibition test (HI).

1. Molecular diagnosis:   detection of viral RNA in plasma or serum or tissues using nucleic acid amplification test (NAAT).  RT-PCR based methods for rapid identification and serotyping of dengue virus in acute phase serum are available.

Interpretation of Dengue Diagnostic Tests:

References and Further Readings:

1. WHO: Dengue Guidelines for Diagnosis, Treatment, Prevention and Control
2. CDC: http://www.cdc.gov/dengue/clinicalLab/laboratory.html