Serologic Tests for Dengue Virus - CDC

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

What is 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 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.

Specimen types

• Serum
• Cerebrospinal fluid (CSF)

Keep in mind

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:
  • Patients with a negative IgM result between days 0–7 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.
• Seroconversion:
  • Seroconversion is observed when an acute sample (usually collected during the first day 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 current dengue. Negative results on both samples are a strong indication the patient was not infected with dengue virus.
  • IgG seroconversion may also be used to diagnose dengue. However, IgG testing is not recommended in a single specimen. The presence of these antibodies may indicate a previous dengue virus infection.
• Cross-reactivity of dengue serological tests:
  • Due to cross-reaction with other flaviviruses and possible nonspecific reactivity, IgM and IgG 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.

Availability

Dengue IgM serologic tests are available as laboratory-developed tests in public health and commercial clinical laboratories. Only one U.S. Food and Drug Administration-cleared dengue IgM detection kit is commercially available. IgG testing is available in some clinical labs. The CDC does not recommend IgG testing on a single specimen for the diagnosis of acute dengue infections.

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Plaque Reduction Neutralization Test (PRNT) and microneutralization tests

What are these tests?

• PRNT is a biological assay based on the virus and antibody interactions. It results in virus inactivation such that the virus is no longer able to infect and replicate in cell culture.
• PRNT can detect and measure the titer of specific neutralizing antibodies against dengue virus and other flaviviruses from the serum of an infected patient.
• 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 is a labor-intensive and relatively costly test.
• A single PRNT test result cannot help determine infection timing. PRNT results from paired acute and convalescent specimens can be used to confirm recent infection.
• 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 should PRNT and microneutralization tests be used?

• PRNT and microneutralization tests can be used when a specific serologic diagnosis is required to rule out Zika or other flaviviruses of concern. For example, 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 flavivirus infections.
• Clinicians should contact local health departments to find out the usefulness and availability of PRNT testing 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 will confirm 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 will confirm a recent infection with that serotype.
• An indication of a recent flavivirus infection is 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.
• 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 a recent flavivirus infection.
• Cases may be re-classified as another flavivirus, such as Zika, according to PRNT results.
• A negative PRNT or microneutralization results on a dengue IgM positive sample during acute illness may indicate no detectable neutralizing activity. An IgM positive result remains presumptive in the absence of other confirmatory tests 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.

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Dengue virus is a flavivirus transmitted to humans by Aedes mosquitoes. An estimated 50-100 million infections occur annually throughout the world. Dengue virus primarily affects persons living in or traveling to tropical areas. Nearly all dengue cases reported in the 48 continental states were acquired elsewhere by travelers or immigrants, with sporadic cases reported along the southern U.S. border (eg, in Arizona, Texas, Florida) and in Hawaii.

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Symptoms of dengue infection can range from mild febrile illness to bleeding (dengue hemorrhagic fever) or hypotension (dengue shock syndrome) with potentially high mortality. It is important to diagnose dengue promptly, since other tropical infections can produce similar patterns of illness and may require different treatment.

Dengue nonstructural protein 1 (NS1) is an antigen produced in abundance by the replicating dengue virus as early as the first day of symptoms and as late as day 18 of symptoms.1 NS1 can be detected in the blood sooner than IgM, which cannot be reliably detected until days 6 to 10.1

Detection of NS1 antigen allows an accurate diagnosis of current infection within the first week of illness (ie, during the febrile phase). Detection of IgM antibody, on the other hand, indicates possible current infection or recent infection (within the past 2 to 3 months).1 An acute and convalescent specimen is thus required to diagnose current infection. Additionally, the NS1 antigen is specific to dengue, whereas the antibody may cross react with other flaviviruses such as West Nile virus, St. Louis and Japanese encephalitis viruses, and yellow fever virus.1

Specimen collection requirements are the same as for the antibody test (ie, 1 mL of room-temperature serum). However, only 1 specimen collection is needed; ie, acute and convalescent specimens are not needed to make the diagnosis.

Yes. The Dengue Fever Antibodies (IgG, IgM) and NS1 Antigen Panel (test code ) includes NS1 antigen and IgM and IgG antibody tests. The panel may be helpful for diagnosing dengue in certain patients. Diagnostic sensitivity during early dengue infection is higher when NS1 is combined with IgM—a significant number of patients will have measurable NS1 but no measurable IgM or IgG during the first week of illness.2,3

NS1 antigen may be ordered separately (test code ). An additional test, Dengue Virus RNA PCR (test code ), may also be useful to detect acute infection in the first five days of illness

There is no specific antiviral therapy currently available to treat dengue infection. Treatment is supportive, based on observation of hemorrhagic and circulatory abnormalities and laboratory test results (eg, electrolytes, platelets and hemoglobin).

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