Dengue Virus - Serology and PCR

Testing Indications

Testing for Dengue virus infection is indicated when a patient displays clinically compatible signs/symptoms of infection and has a compatible exposure history (e.g. returned from travel to a Dengue endemic area, noted mosquito bites, among others).

Following exposure to the Dengue virus (e.g. after a bite from an infected mosquito), individuals may remain asymptomatic or may develop an acute febrile illness within 3 to 14 days.² This primary infection can be associated with mild symptoms, including but not limited to headache, myalgia/arthralgia, nausea and vomiting or a rash. In some cases, this primary infection, or more commonly a secondary Dengue virus infection at a later time, may progress to a more severe form with hemorrhagic manifestations, abdominal pain and vomiting and can culminate with shock and organ failure.

Laboratory diagnosis can be accomplished by testing serum or plasma to detect viral nucleic acid or virus specific antibodies. Testing by PCR can be performed during the viremic phase of infection which is typically within the first 7 days after symptom onset⁴, while serology testing (IgM and IgG) can be performed from approximately 7 days from symptom onset⁵. Testing of asymptomatic individuals is not recommended.

Acceptance/Rejection Criteria
Specimens received without the appropriate forms (See: Submission and Collection Notes) will not be tested.

Timing of Specimen Collection

Serology:
Acute and convalescent sera should be collected for serologic testing, where applicable. The convalescent serum specimen should be collected at least 2 to 3 weeks after the initial acute specimen.

Molecular Real-Time PCR:
Clotted blood, serum or plasma specimens for PCR testing should be collected as soon as possible after symptom onset, but no later than 14 days following onset of illness.

If CSF is submitted for suspected CNS infection, collect as soon as possible.

Limitations

Hemolysed, icteric, lipemic or microbial contaminated sera or plasma are not recommended for testing.

Storage and Transport

• For serum separator tubes: centrifuge specimens prior to placing in biohazard bag.
• Place each specimen type in an individual biohazard bag and seal. Insert the corresponding requisition in the pocket on the outside of each sealed biohazard bag.
• Clotted blood/serum should be stored at 2-8°C following collection and shipped to PHO on ice packs.
• For any other specimens submitted for molecular testing, specimens may be stored at 2-8°C following collection and shipped to PHO on ice packs, but should be frozen (at -80°C) and shipped on dry ice if delivery to PHO will take more than 72 hours.

Special Instructions

• Each specimen submitted for testing must be accompanied by a separate General Test Requisition. All fields on each requisition must be completed.
• It is mandatory to submit the Arbovirus (Non-Zika) Testing Intake Form, if PCR requested, with all fields completed. However, if all of the requested information from the Arbovirus (Non-Zika) Intake Form has been provided on the General Test Requisition, the additional Arbovirus (Non-Zika) Testing Intake Form is not required for tests that do not require PHO Microbiologist approval. Specimens submitted with this mandatory information missing will not be tested until it’s provided.

Test Frequency and Turnaround Time (TAT)

Serology testing TAT is up to 8 days from the receipt at PHO.

Molecular testing TAT is up to 5 days for PHO test results. If supplementary testing at the National Microbiology Laboratory is required, TAT may be up to 21 days.

Serology:
The Dengue IgM & IgG test is performed using a commercial ELISA (Enzyme Linked Immunosorbent Assay) test kit.

Molecular Real-Time PCR:
Molecular testing is conducted using a laboratory developed real-time polymerase chain reaction (RT-PCR) panel which simultaneously tests for genomic targets of the Zika, Chikungunya and Dengue viruses. All four serogroups of Dengue virus (1 to 4) can be detected, but are not distinguished by this assay. Specimens submitted for testing of any of the three virus targets will be tested using the Arbovirus panel and all three virus results will be reported.

Interpretation

Serology:
The Anti-Dengue Virus ELISA (IgM and IgG) is suitable for the serological detection of an acute or a past dengue virus infection and supplements direct pathogen detection. Seroconversion in the IgM or IgG antibodies alongside compatible symptoms may indicate an acute or recent infection. Flavirivus IgM antibodies may persist for several months following disease onset and reactivity in this setting may reflect a recent or resolved infection. Reactive serology results may also occur following Dengue vaccination. Consult the table below for interpretations of Dengue virus serologic testing.

Serologic testing for Dengue virus is limited by cross-reactions that may occur with other related Flaviviruses (e.g. St. Louis Encephalitis, West Nile, Japanese B Encephalitis, Powassan and Yellow Fever viruses). Serology results should be interpreted in the context of the clinical and travel history of the patient. A non-reactive serological result does not rule out Dengue virus infection.

Molecular Real-Time PCR:
A positive PCR result (Dengue virus detected by RT-PCR) indicates that Dengue virus nucleic acids were detected in the specimen and an acute infection.

A negative PCR result (Dengue virus not detected by RT-PCR) indicates that Dengue virus nucleic acids were not detected in the specimen. This does not exclude Dengue virus infection.

Reporting

Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

[For reportable diseases: Positive IgM and RT-PCR specimens from patients with Viral hemorrhagic fever are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.]²

References

1. Dengue fever
2. Dengue
3. https://www.simcoemuskokahealth.org/docs/default-source/jfy-health-care-professionals/reportable-diseases/hemorrhagic-fevers.pdf?sfvrsn=2
4. Molecular Tests for Dengue Virus.
5. Serologic Tests for Dengue Virus
6. Pabbaraju et al. 2016. Simultaneous detection of Zika, Chikungunya and Dengue viruses by a multiplex real-time RT-PCR assay. J. Clin. Virol. 83:66-71.