Serologic Tests for Dengue Virus
June 14, 2019, Morbidity and Mortality Weekly Report: Dengue and Zika virus diagnostic testing for patients with a clinically compatible illness and risk for infection with both viruses
Dengue virus-specific IgM and neutralizing antibodies typically develop toward the end of the first week of illness. IgM levels are variable, but generally are positive starting 4-5 days after onset of symptoms and continuing for approximately 12 weeks post symptom onset, but may persist longer.
IgM Antibody Capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA)
What is the test?
- The dengue MAC-ELISA is used for the qualitative detection of dengue virus IgM antibodies.
- 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 should it be used and at what time during infection?
- As the immune system fights the infection, IgM antibodies against dengue virus are detectable starting 4-5 days after onset of symptoms and are reliably detectable for approximately 12 weeks.
- Combined testing with a nucleic acid amplification test (NAAT) and MAC-ELISA usually provides a diagnostic result during the first 1-7 days of illness. A convalescent phase specimen is needed to make a diagnosis of dengue virus infection when results are negative on both tests from the acute specimen.
- IgM antibody detection is not useful for dengue virus serotype determination.
- Cerebrospinal fluid
*Some IgM tests can be performed on plasma and whole blood but these tests have not been extensively evaluated for these specimen types.
Interpretation of results
- Positive IgM: Patients with a positive IgM test result are classified as presumptive, recent dengue virus infections.
- Negative IgM:
- Patients with negative IgM results before day 8 of illness and absent or negative NAAT or NS1 results are considered unconfirmed cases. For these cases, a second sample should be obtained after day 7 of symptoms for additional serologic testing.
- Patients with negative IgM results after 7 days of symptoms, and absent or negative NAAT or NS1 (dengue virus antigen detection) are classified as negative for recent infection.
- Patients with a change from negative to positive IgM results in paired samples (first sample collected during the first 7 days of illness, and second sample collected after symptoms subside) are classified as current dengue infections.
- Due to cross-reaction with other flaviviruses and possible nonspecific reactivity, results may be difficult to interpret. Consequently, presumed positive, indeterminate, and equivocal, IgM antibody test results may be forwarded for confirmation by plaque reduction neutralization testing (PRNT), see below.
One US Food and Drug Administration-cleared serologic dengue IgM detection kit is commercially available. Dengue IgM serologic tests also are available as laboratory-developed tests in public health and commercial clinical laboratories or as diagnostic kits.
Plaque Reduction Neutralization Test (PRNT)
What is the test?
- PRNT can detect specific neutralizing antibodies against dengue virus and other flaviviruses.
- PRNT is used to more precisely 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 yellow fever).
- PRNT measures the titer of the neutralizing antibodies in the serum of the infected person.
- PRNT is a biological assay based on the principle of interaction of virus and antibody resulting in inactivation of virus such that the virus is no longer able to infect and replicate in cell culture.
- The microneutralization assay is based on the same principle as the PRNT, however instead of counting the number of plaques per well, the assay uses a colorimetric or fluorometric measurement of numbers of infected cells to determine the end-point dilution. This assay was developed to use less reagents and for testing a larger number of samples.
- PRNT is a labor-intensive and relatively costly test.
- A single PRNT test result cannot help determine the timing of infection.
How should PRNT be used and at what time during infection?
- PRNT and microneutralization PRNT can be used when a specific serologic diagnosis is required, for example in pregnant women or cases of clinical and epidemiologic importance.
- PRNT is used in an attempt to confirm the infecting virus in a dengue or Zika virus IgM-positive specimen and can in some cases 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, PRNTs may have less utility in differentiating flavivirus infections. In resource-limited countries, PRNTs may not be available. Clinicians should contact local health departments to find out the usefulness and availability of PRNT testing as a confirmatory test for IgM positive cases. For example, PRNT testing is not routinely performed in the US territory of Puerto Rico where dengue is endemic and Zika virus has circulated. However, strong consideration should be given to performing PRNT in symptomatic pregnant women with negative NAAT testing and positive IgM serology. Another situation in which PRNT testing would not likely add value is in the context of a documented dengue outbreak in an area without evidence of concurrently circulating flaviviruses.
- Serum, CSF
Interpretation of results
- PRNT performed on a dengue IgM-positive specimen showing neutralizing antibodies to only one dengue virus serotype confirms 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 a recent infection with that serotype.
- A 4-fold rise in PRNT titers in paired acute and convalescent samples with antibodies to multiple dengue virus serotypes or other flaviviruses in the convalescent sample is classified as a recent flavivirus infection.
- PRNT performed on a single dengue IgM-positive specimen may show neutralizing antibody titers for dengue, Zika, and other flaviviruses included in the test. These results are classified as recent flavivirus infection.
- Cases may be re-classified as another flavivirus, such as Zika, according to PRNT results.
- A negative PRNT result on a dengue IgM positive sample indicates a false positive result, indicating no evidence of infection.
- A suspected case cannot be confirmed if there is no evidence of a flavivirus infection through PRNT.,.
PRNT is 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.