Dengue Virus - Serology and PCR

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Background
This page provides information on the testing available for Dengue virus at Public Health Ontario. The Dengue virus is a single-stranded RNA virus that is a member of the Flaviviridae family and can cause a febrile illness known as Dengue fever. The primary route of transmission for this virus is though the bite of infected mosquitos in tropical and subtropical areas. Testing for Dengue virus infection can be performed by serology or PCR depending on the specific clinical scenario.

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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.

Specimen Collection and Handling

Specimen Requirements

Test Requested

Required Requisition(s)

Specimen Type

Minimum Volume

Collection Kit

Dengue Virus serology

General Test Requisition

Blood, serum or plasma

5 ml blood or 1 ml serum

Serum separator tubes (SST)

EDTA, heparin or citrated plasma

Dengue Virus PCR

General Test Requisition Arbovirus (Non-Zika) Testing Intake Form

Blood, serum or plasma

1.0 ml

Serum separator tubes (SST)

EDTA, heparin or citrated plasma

Submission and Collection Notes

Pregnancy status, mosquito exposure, patient symptoms, travel history to endemic areas (including dates of departure and return) and date of collection of specimens (within 14 days of symptom onset date for PCR testing) should be clearly indicated on the requisition.

Testing is not validated for specimen types not included in the table above (e.g. CSF) and must be pre-approved by a PHO Microbiologist. Please contact PHO Laboratory Customer Service Centre at 416-235-6556 or 1-877-604-4567 before submission.

If Dengue PCR is requested, the Arbovirus (Non-Zika) Testing Intake Form is a mandatory requirement for testing. PHO utilizes the information on the requisition and the mandatory intake form to assess testing criteria, assign appropriate tests, and provide mandatory information required by the National Microbiology Laboratory

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.

Requisitions and Kit Ordering

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.

Test Methods

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.

IgM ELISA	IgG ELISA	Reported Results	Comments
Non-reactive	Non-reactive	No serological evidence of infection.
Indeterminate	Indeterminate	Antibody status inconclusive.	Advise a follow up in 7 to 14 days if clinically indicated
Non-reactive	Indeterminate	Antibody status inconclusive.	Advise a follow up in 7 to 14 days if clinically indicated
Reactive	Non-reactive or Indeterminate	May indicate recent infection.	Advise a follow up in 7 to 14 days if clinically indicated
Indeterminate	Non-reactive	Antibody status inconclusive.	Advise a follow up in 7 to 14 days if clinically indicated
Non-reactive	Reactive	No serological evidence of recent Dengue infection. Evidence of previous Flavivirus infection.
Indeterminate	Reactive	Antibody status inconclusive.	Advise a follow up in 7 to 14 days if clinically indicated
Reactive	Reactive	Indicates recent primary 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

Dengue fever
Dengue
https://www.simcoemuskokahealth.org/docs/default-source/jfy-health-care-professionals/reportable-diseases/hemorrhagic-fevers.pdf?sfvrsn=2
Molecular Tests for Dengue Virus.
Serologic Tests for Dengue Virus
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.