Serologic Tests for Dengue Virus

IgM Antibody Capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA)

Description of the test

• The dengue MAC-ELISA is used for the qualitative detection of IgM antibodies against dengue virus.
• The MAC-ELISA is based on capturing human IgM antibodies on a microtiter plate using anti-human-IgM antibody, followed by the addition of dengue virus antigens. The antigens used for this assay are derived from the envelope proteins of the four dengue virus serotypes (DENV-1-4).

How and when to use the test

• As the immune system fights the infection, IgM antibodies against dengue virus are detectable starting 4–5 days after onset of symptoms and for up to 3 months.
• Combined testing with a nucleic acid amplification test (NAAT) and an IgM antibody test usually provides a diagnostic result during the first 1-7 of illness. A second specimen collected >7 days from symptom onset can support a diagnosis of dengue virus infection when the acute specimen test results are negative or unavailable.
• IgM antibody detection is not useful for dengue virus serotype determination.

Specimen types

• Serum (preferred)
• Cerebrospinal fluid (CSF)

Interpretation of results

• Positive IgM: Patients with a positive IgM test result in a single sample are classified as presumptive, recent dengue virus infections.
• Negative IgM:
  • During the first 7 days after symptom onset, negative results by IgM do not rule out the diagnosis of dengue. Testing in combination with dengue NAAT or NS1 is recommended during this period to help confirm infection.
    
  • Patients with negative IgM results more than 7 days after symptom onset are considered negative for recent dengue virus infection.
    
• Seroconversion:
  • Collecting paired samples, at least 7 days apart can help identify or rule out recent dengue virus infection. For example, collect one sample during the acute phase (within the first 7 days) and collect a second sample during the convalescent phase (after 7 days).
  • Seroconversion is observed when an acute sample (usually collected during the first 7 days of illness), and a convalescent sample (collected after 7 days), are tested for IgM. If test results change from negative to positive, the patient is diagnosed with recent dengue infection. Negative results on both samples provide strong evidence that the patient was not infected with dengue virus.
    
• Cross-reactivity of dengue serological tests:
  
  • Due to potential cross-reactivity with other flaviviruses, such as Zika and West Nile virus, IgM and IgG results may be difficult to interpret. Positive, indeterminate, and equivocal IgM antibody test results may require confirmation by plaque reduction neutralization testing (PRNT) to differentiate between flaviviruses. This is particularly important if a dengue infection is identified in an area without known dengue transmission, or in cases where a specific diagnosis is clinically or epidemiologically significant.

Availability

Dengue IgM serologic tests are available in public health and commercial clinical laboratories. Currently, only one dengue IgM detection kit is cleared by the U.S. Food and Drug Administration and commercially available. IgG testing is available in some clinical labs; however, the CDC does not recommend IgG testing on a single specimen for the diagnosis of acute dengue virus infection.

IgG Testing

• These tests detect antibodies from past infections with dengue or other flaviviruses.
• Serologic testing by IgG ELISA in a single specimen is not recommended for diagnosis of acute dengue in patients.
• IgG seroconversion occurs when a person's IgG results change from negative to positive in paired samples, usually taken at least 14 days apart. A greater than 4-fold rise in IgG titers between two samples can also indicate a recent infection.
• The presence of IgG antibodies alone does not confirm recent infection, as individuals with secondary dengue infections may already have IgG due to previous exposure, and cross-reactivity with antibodies from other flavivirus infections can lead to positive results.

Plaque Reduction Neutralization Test (PRNT) and microneutralization tests

Description of these tests

• PRNT is a laboratory assay based on the interactions between the virus and antibodies. It involves exposing cells in a laboratory setting to the dengue virus. If neutralizing antibodies are present in the blood sample being tested, they will reduce the number of "plaques" (areas of infected cells) that form on the cell culture.
• PRNT can measure virus-specific neutralizing antibodies titers against dengue virus and other flaviviruses from the serum of a current or previously infected patient.
• PRNT can be used to determine the cause of infection in IgM-positive patients for whom a specific diagnosis is clinically or epidemiologically important (e.g., to rule out other flaviviruses such as Zika or West Nile virus).
• A single PRNT result cannot determine the timing of infection because the presence of neutralizing antibodies may be due to a recent infection, a past infection, or vaccination.
• PRNT results should be interpreted in conjunction with IgM results and previous exposure history, and ideally in paired acute and convalescent specimens.
• The microneutralization assay is based on the same principle as the PRNT.
  • The assay uses a colorimetric or fluorometric measurement of numbers of infected cells.
  • This assay was developed to use less reagents and for testing a larger number of samples.

How and when to use these tests

• PRNT and microneutralization tests can be used when a specific serologic diagnosis is required to rule out Zika or other flaviviruses of concern (e.g., in pregnant women or in cases of clinical and epidemiologic importance).
• These tests are used to confirm the infecting virus in a dengue or Zika virus IgM-positive specimen. In some cases, it can determine the infecting dengue virus serotype.
• For residents living in areas with dengue and Zika virus circulation, or for travelers with multiple long-term exposures, PRNT and microneutralization tests may have less utility in differentiating between current and past flavivirus infections.
• Clinicians should contact local health departments to consider PRNT as a confirmatory test for IgM positive cases.

Specimen types

• Serum
• CSF

Interpretation of results

• PRNT performed on a dengue IgM-positive specimen showing neutralizing antibodies to only one dengue virus serotype confirms recent infection with that serotype.
• A seroconversion from a negative PRNT in an acute sample to a positive PRNT with antibodies to only one dengue virus serotype in a convalescent sample confirms recent infection with that serotype.
• A 4-fold rise in PRNT titers in paired samples with antibodies to a dengue virus indicates a recent infection to that serotype.
• A 4-fold rise in PRNT titers in paired samples with antibodies to multiple flaviviruses indicates a recent flavivirus infection.
• PRNT performed on a dengue IgM-positive specimen showing neutralizing antibody titers to multiple flaviviruses (ex: dengue and Zika) are classified as a recent flavivirus infection.
• Cases may be re-classified as another flavivirus (ex: Zika) based on PRNT results.
• A negative PRNT or microneutralization results on a dengue IgM positive sample during acute illness may indicate no detectable neutralizing activity. Collecting a convalescent specimen for testing by IgM and PRNT can help confirm or rule out infection in these cases. An IgM positive result remains presumptive in the absence of other confirmatory test results (PRNT, microneutralization tests, NS1, or NAAT).

Availability

PRNT and microneutralization tests are performed at CDC or a laboratory designated by CDC (i.e., a laboratory that has independently demonstrated proficiency to perform PRNT testing by completing a proficiency panel provided by CDC).

Public health laboratories may refer IgM-positive specimens to CDC for PRNT to confirm positive, equivocal, or inconclusive IgM results. In some resource limited countries, PRNTs and microneutralization tests may not be available.