Yellow Fever Virus – Serology and PCR

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Background
This page provides information on the testing available through Public Health Ontario (PHO) for the Yellow Fever virus (YFV).

The YFV is a mosquito-borne RNA virus that is a member of the Flaviviridae family.
The primary route of transmission of this virus is through the bite of an infected mosquito¹, typically in tropical regions of South America and Africa^2,3.

Testing for Yellow Fever involves serology and/or polymerase chain reaction (PCR) depending on the specific clinical scenario. Appropriate specimens that are received by PHO are referred to the National Microbiology Laboratory (NML) in Winnipeg for testing.

Updates
Effective July 2, 2025, submission of the new Vector-borne and Zoonotic Virus Testing Intake Form is mandatory, along with the General Test Requisition when requesting specific vector-borne or zoonotic virus tests. The new intake form replaces both the Arbovirus (Non-Zika) Testing Intake Form and the Mandatory Intake Form for Zika Virus Testing.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 14 juill. 2025.

Testing Indications

Note: A clinical and epidemiological risk assessment for YFV infection is recommended prior to submitting a YFV test request.

Testing for Yellow Fever virus infection is indicated for individuals with:

clinically compatible signs/symptoms of infection and
relevant exposure history (e.g. travel to an endemic area, noted mosquito bites during travel, among others)
no history of vaccination against YFV^4,5 OR new onset of compatible symptoms following recent YFV vaccination

Most YFV infections are asymptomatic, however, some individuals, (e.g. those without prior immunization), can develop YFV disease. Symptoms can develop within 3 to 6 days of exposure and include, but are not limited to, fever, malaise, headache, photophobia, myalgias/arthralgia, or a rash^1,6. More severe disease manifestations can occur, characterized by jaundice, abdominal pain and hepatitis, with hemorrhagic manifestations also possible.

Molecular testing may be used to detect the virus during the acute phase of the infection in individuals suspected of an active infection. Serologic testing is indicated for individuals with prolonged symptoms or those suspected of having a resolved infection. Testing is not recommended for asymptomatic individuals unless confirming titres for those that are at high-risk of infection (e.g. ongoing risk of exposure, among others)¹.

Acceptance/Rejection Criteria

Specimens received without the appropriate forms (See: Submission and Collection Notes) are subject to cancellation.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Yellow Fever Virus serology	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum	5.0 ml blood or 1.0 ml serum	Red top or Serum Separator Tube (SST)
Yellow Fever Virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum	5.0 ml blood or 1.0 ml serum	Red top or Serum Separator Tube (SST)
Yellow Fever Virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum	1.0 ml	Plasma with non-heparin anticoagulant
Yellow Fever Virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	CSF	400 µl	Sterile container

Submission and Collection Notes

Label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. For additional information see: : Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay.

Each specimen submitted for testing must be accompanied by a separate PHOGeneral Test Requisition, with all fields completed.

It is MANDATORY to provide the clinical information, relevant travel(s), and relevant exposures for Vector-borne viruses requested on the Vector-borne and Zoonotic Virus Testing Intake Form. Test requests that are submitted without the appropriate mandatory information are subject to cancellation.

If submitting CSF, a paired serum specimen must also be submitted.

Testing for Yellow Fever Virus PCR is not routinely performed and must be approved by a PHO Microbiologist. Submission of the mandatory Vector-borne and Zoonotic Virus Testing Intake Form will initiate the review process at PHO provided the form contains all necessary information. Requests received without the form, forms submitted with insufficient information or insufficient justification for testing are subject to cancellation.

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 RT-PCR):
Specimens for molecular testing (PCR) should be collected as soon as possible after the onset of symptoms, unless otherwise indicated via discussion with a PHO Microbiologist.

Limitations

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

Storage and Transport

All clinical specimens must be shipped in accordance with the Transportation of Dangerous Goods Act/Regulations.

For serum separator tubes: centrifuge sample 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 /CSF specimens should be stored at 2-8°C following collection and shipped to PHO on ice packs.

All specimens submitted for molecular testing should be stored at 2-8°C following collection and shipped to PHO on ice packs. If a delay in transport to PHO is anticipated (more than 72 hours), specimens should be frozen (at -80°C if possible) and shipped on dry ice.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

This is a Referred Out Test. Specimens are sent to the NML in Winnipeg for testing. Testing is not performed at PHO.

The TAT for YFV serology and molecular testing is up to 28 days from receipt at PHO. The YFV RT-PCR is not performed routinely.

Test Methods

Serology is performed at the NML by Plaque Reduction Neutralization Test (PRNT).

Molecular testing is performed by RT-PCR at the NML for investigational use only⁷. This test requires prior approval by the PHO Microbiologist.

Interpretation

All results should be interpreted in the context of the specific clinical scenario. Given the overlap in distribution of disease vectors, testing for other potential pathogens should be considered, where applicable.

Serology:
A ≥4-fold increase in neutralizing antibody titre between acute and convalescent sera (collected 2 to 3 weeks apart) tested by PRNT is considered indicative of seropositivity.

Additional notes on YFV serology:

Cross-reactivity (a reactive test result) may occur with other Flaviviruses such as Dengue virus, West Nile virus; therefore, submitting both acute and convalescent sera is recommended.
Testing for other vector-borne viruses may be added by the NML to assist with interpretation.

Molecular (Real-Time PCR):
A positive PCR result indicates that YFV nucleic acids were detected in the specimen and an acute/recent infection.

A negative PCR result indicates that YFV nucleic acids were not detected in the specimen. This does not exclude YFV infection.

Reporting

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

References

Public Health Agency of Canada. Yellow Fever [Internet]. Ottawa: Public Health Agency of Canada; [cited 2023]. Available from: https://www.canada.ca/en/public-health/services/diseases/yellow-fever.html
Centers for Disease Control and Prevention. 2025. Countries at Risk for Yellow Fever: Africa. Available from: https://www.cdc.gov/yellow-fever/africa/index.html
Centers for Disease Control and Prevention. 2025. Countries at Risk for Yellow Fever: South America. Available from: https://www.cdc.gov/yellow-fever/south-america/index.html
World Health Organization. 2022. Countries with risk of yellow fever transmission and countries requiring yellow fever vaccination (November 2022). Available from: https://www.who.int/publications/m/item/countries-with-risk-of-yellow-fever-transmission-and-countries-requiring-yellow-fever-vaccination-(november-2022)
Public Health Agency of Canada. Canadian Immunization Guide – Part 4: Active Vaccines [Internet]. Ottawa: Public Health Agency of Canada; [cited 2023]. Available from: https://www.canada.ca/en/public-health/services/publications/healthy-living/canadian-immunization-guide-part-4-active-vaccines/page-25-yellow-fever-vaccine.html
World Health Organization. Yellow fever [Internet]. Geneva: World Health Organization [cited 2023]. Available from: https://www.who.int/news-room/fact-sheets/detail/yellow-fever
(Yellow fever (who.int))
National Microbiology Laboratory. 2025. Molecular detection of Yellow Fever Virus by real-time reverse transcriptase PCR (RT-PCR). Available from: https://cnphi.canada.ca/gts/reference-diagnostic-test/5536?alphaReturn=pathogenByLetter&alphaChar=Y

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 14 juill. 2025.

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Mis à jour le 2 juill. 2025