Respiratory Viruses (including influenza)

Conformément au Règlement de l’Ontario 671/92 de la Loi sur les services en français, les renseignements d’analyses de laboratoire liés à la présente page ne sont offerts qu’en anglais parce qu’ils sont de nature scientifique ou technique et destinés uniquement à l’usage des fournisseurs de soins de santé qualifiés et non aux membres du public.

Background
This page provides information on molecular testing for respiratory viruses at Public Health Ontario (PHO).

PHO utilizes a testing algorithm for influenza and other respiratory viruses that is based on patient settings.

For information on influenza A subtyping conducted under PHO’s enhanced influenza surveillance program, involving specimens that tested positive for influenza A at non-PHO external laboratories, please refer to the Avian Influenza TIS.

For information regarding other testing options, refer to the following PHO webpages:

Updates

As of April 1, 2025, molecular respiratory test panels will be conducted at Toronto, London, Ottawa, or Timmins laboratory site. Please refer to the updated Table 2: Respiratory virus testing conducted at PHO locations under Algorithm section.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 01 mai 2025.

Testing Indications

PHO provides testing using 2 different respiratory test panels – these are multiplex respiratory virus PCR (MRVP) and FLUVID

MRVP testing is performed on the following eligible persons:

Symptomatic children (<18 years) seen in the Emergency Department (ED)
Symptomatic hospitalized patients (ward, inpatient and ICU/CCU)
Symptomatic residents in institutional settings (non-outbreak)
Specimens from the first four symptomatic patients/residents in an outbreak setting that request respiratory virus testing

FLUVID testing is performed on the following eligible persons:

Symptomatic healthcare workers/staff in the institutional setting in an outbreak setting requesting COVID-19 and respiratory virus testing or residents after the first four have already been tested for COVID-19 and MRVP
Symptomatic adult individuals seen in the Emergency Department (ED) who are at risk for severe illness/outcome and for whom care or treatment decisions may be impacted by test results

Respiratory virus test requests for patients with acute respiratory illness (ARI) in the settings described above should be clearly indicated on the COVID-19 and Respiratory Virus Test Requisition by selecting “Respiratory Viruses” or “COVID-19 Virus AND Respiratory Viruses” as appropriate in section “5 – Test(s) Requested” on the requisition. Only select one of the three test request options. SEE NOTE #1 below.

In addition, the patient’s setting and symptoms must be clearly indicated on the requisition.

NOTE: See “TEST ALGORITHM AND METHODS” Table footnote 3 for FLUVID targets and footnote 4 for MRVP targets.

Acceptance/Rejection Criteria

Label the specimen container with:

The patient’s first AND last name
Date of collection
One other unique identifier, such as the patient’s date of birth or Health Card Number

See Criteria for Acceptance of Patient Specimens.

Asymptomatic patients (or requisitions with no symptoms checked off) and requisitions lacking patient setting will not be tested for respiratory viruses.

Specimens collected using expired swabs or transport media tubes will be rejected.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Respiratory Virus (Molecular)	COVID-19 and Respiratory Virus Test Requisition	Upper respiratory tract: Nasopharyngeal swab (NPS)	Nasopharyngeal swab in universal transport media (UTM)	Virus-Respiratory Collection Kit: order# 390082
Respiratory Virus (Molecular)	COVID-19 and Respiratory Virus Test Requisition	Viral throat swab (see Submission and Collection Notes below)	Swab in universal transport media (UTM)	Virus PCR Kit order#: order# 390081
Respiratory Virus (Molecular)	COVID-19 and Respiratory Virus Test Requisition	Lower respiratory tract (when possible): sputum, BAL, bronch wash, pleural fluid, lung tissue, tracheal aspirate (see Submission and Collection Notes below)	1.0 ml (if applicable)	Sterile container; Tuberculosis Kit order#: 390042

Submission and Collection Notes

To request respiratory virus testing only, check the “Respiratory Viruses” box in Section 5 of the COVID-19 and Respiratory Virus Test Requisition (the preferred requisition for COVID-19 and seasonal respiratory virus testing).

To request COVID-19 AND respiratory virus testing, check the “COVID-19 Virus AND Respiratory Virus” box in Section 5 of the requisition.

Only check one of the three boxes.

NPS is the most sensitive specimen type for respiratory virus testing. Apart from neat saliva and mouth rinse/swish and gargle, these collection kits and associated specimen sources can also be used for seasonal respiratory testing (e.g. MRVP) provided suitability criteria are met, if the specimen is also being tested for COVID-19.

Note: Saliva and mouth rinse/swish and gargle specimens have not been validated for seasonal respiratory virus testing at PHO and will not be accepted for testing respiratory viruses other than SARS-CoV-2.

Complete all fields of the COVID-19 Test Requisition. Ensure the following fields are completed if diagnostic testing is requested:

Patient setting (mandatory)*
Symptoms (indicate all symptoms) (mandatory)*
Medical condition that puts the patient at increased risk for severe illness (mandatory if applicable)*
Valid Outbreak/Investigation Number (if applicable)*
Onset date
Requesting healthcare provider’s CPSO#,
Complete mailing address, telephone, and fax number
Patient’s full name and address, date of birth, health card number (must match the specimen label)

*MANDATORY INFORMATION IS USED FOR TRIAGING SPECIMENS. FAILURE TO PROVIDE THIS INFORMATION MAY RESULT IN REJECTION OR TESTING DELAY.

Cytomegalovirus (CMV) PCR is available upon request (indicate on the requisition) for Broncho alveolar lavage (BAL)/bronchial wash/pleural fluid.

Respiratory outbreak specimens may be submitted following the Respiratory Outbreak Testing Prioritization Protocol.

Check the expiry dates of both the collection swabs and transport media (tube), as specimens collected using expired swabs or tubes will be rejected. In addition, specimens must meet the Criteria for Acceptance of Patient Specimens.

NOTE: “Molecular transport media” (media containing guanidine) is unsuitable for influenza rapid testing. Specimens which are not suitable for rapid testing will not be tested by rapid test and will be sent directly for MRVP or FLUVID testing.

Storage and Transport

Place the specimen container in the biohazard bag and seal the bag; insert the completed requisition in the pocket on the outside of the sealed biohazard bag. To maintain optimal specimen integrity, specimens should be stored at 2-8°C following collection and should be transported to PHO on ice packs within 72 hours of collection. If longer storage/transit time is anticipated, specimens should be frozen at -70°C or lower and transported on dry ice.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

The TAT for MRVP testing is up to 4 days after receipt by PHO.
FLUVID testing is performed 7 days a week.

TAT: 48 hours from receipt at PHO.
Turnaround time may vary according to geographical location and proximity to PHO.

NOTE: When performed, TAT for the Direct Antigen Test for Influenza (Influenza rapid) is within 1 day after receipt at any PHO location.

Test Methods

FLUVID – FLUVID Multiplex (MPX) PCR:

Laboratory developed test (LDT) validated at PHO
Detects influenza A, influenza B, RSV (A/B) and SARS CoV-2 (COVID-19) by multiplex nucleic acid amplification testing (NAAT)– see note 3, below

MRVP – Respiratory In House (INH) MPX PCR:

Laboratory developed test (LDT) validated at PHO
Detects respiratory targets by multiplex NAAT – see note 4 below

Algorithm

PHO utilizes a testing algorithm for influenza and other seasonal respiratory viruses that is based on patient setting.

The Eligibility criteria for COVID-19 and Influenza Testing for Patients at Risk of Severe Illness has been updated to align with the current guidelines from Ministry of Health and Public Health Agency of Canada.

Respiratory virus testing is available according to the following tables:

Table 1: Eligibility criteria for respiratory virus testing available at PHO for symptomatic (ARI) patients by patient setting and outbreak status

Patient Setting¹	Testing Available By Request
Hospitalized (all inpatients)²	SARS-CoV-2 and MRVP^4,5 OR FLUVID³ followed by MRVP^4,5 (Both combinations will provide testing for the same viruses.)
Remote communities	SARS-CoV-2 and MRVP^4,5 OR FLUVID³ followed by MRVP^4,5 (Both combinations wil provide testing for the same viruses.)
Public health unit declared respiratory infection outbreaks (including institutional outbreaks e.g. long-term care homes, correctional facilities, congregate living settings) – residents only	SARS-CoV-2 and MRVP^4,5 OR FLUVID³ and MRVP^4,5 MRVP for up to 4 outbreak related specimens⁶ Additional specimens from symptomatic individuals will be tested by FLUVID only.⁵ Influenza rapid testing⁷ (will be done if PCR testing is delayed >24 hours )
Public health unit declared respiratory infection outbreaks (including institutional outbreaks e.g. long-term care homes, correctional facilities, congregate living settings) – healthcare workers/staff	FLUVID³
Institutions (non-outbreak) (e.g. long-term care homes, correctional facilities, congregate living settings)¹⁰ – residents only	SARS-CoV-2 and MRVP^4,5 OR FLUVID³and MRVP^4,5
Emergency Department (paediatric patients <18 years old)¹¹	SARS-CoV-2 and MRVP^4,5 OR FLUVID³ and MRVP^4,5
Emergency Department (adult patients) for individuals at risk of severe illness or outcome in whom care or treatment decisions may be impacted by test results	FLUVID³
Ambulatory settings and meets Ministry guidance criteria for COVID-19 testing,⁹	SARS-CoV-2^1,9

Table footnotes:

The specific test being requested AND patient setting must appear on the requisition to help with appropriate test assignment and triaging of specimens. If patient setting is not provided, the specimen will not be tested for COVID-19 or Respiratory viruses. For outbreaks or investigations, the requisition must include the assigned valid outbreak or investigation number.
All ICU (Intensive Care Unit)/ CCU (Critical Care Unit) and ward inpatients.
FLUVID detects: influenza A, influenza B, respiratory syncytial virus (RSV A/B), and SARS-CoV-2 (COVID-19). This assay may be used as an initial test prior to MRVP to provide earlier results during influenza and RSV seasons.
MRVP detects: influenza A, influenza A H3 subtype, influenza A H1 (pdm09) subtype, influenza B, respiratory syncytial virus (RSV A/B), parainfluenza (1 – 4), adenovirus, enterovirus, seasonal human coronavirus (OC43, 229E, NL63, HKU1), rhinovirus and human metapneumovirus.
Note: The assay detects the different RSV, parainfluenza and seasonal human coronaviruses named above but does not differentiate between them. It does not detect or cross-react with SARS-CoV-2.
A limit of four outbreak specimens are routinely accepted for MRVP testing if ordered on PHO’s requisition. Additional specimens from symptomatic patients will be tested using FLUVID test. For requests for additional MRVP testing in outbreak settings, contact PHO’s Customer Service Centre. Respiratory outbreak specimens may be submitted following the Respiratory Outbreak Testing Prioritization Protocol
Caution: influenza rapid testing can only be performed if the specimen is received in suitable media. See Submission and Collection Notes above.
Influenza rapid antigen testing will be performed on the first four outbreak specimens from symptomatic patients if influenza PCR testing (e.g. MRVP, FLUVID) commencement is delayed to >24 hours from specimen receipt at PHO.
To assist with patient management when respiratory virus testing is not available, healthcare providers are encouraged to refer to Ontario Respiratory Virus Tool for information on respiratory pathogen activity in Ontario.
Respiratory virus testing is not offered to ambulatory patients seen in outpatient setting, clinics. SARS-CoV-2 testing is offered to those who meet testing criteria as indicated in the Ministry of Health’s current guidelines.
Schools and daycare centres are not considered institutions for the purpose of sporadic MRVP testing.
Specimens collected from paediatric patients (<18 years old) seen in the Emergency Department (ED) will be accepted for MRVP testing to support enhanced respiratory virus surveillance.

Table 2: Respiratory virus testing conducted at PHO locations:

PHO Locations	Testing conducted	Management of specimens
Toronto, London, Ottawa, Timmins	Rapid Flu, SARS-CoV-2, FLUVID, MRVP	Specimens will be tested at receiving site
Hamilton, Kingston, Thunder Bay, Peterborough, Orillia, Sault Ste. Marie, Sudbury	Rapid Flu, no molecular respiratory testing	Specimens transferred to one of the above laboratory sites if MRVP, FLUVID or SARS-CoV-2 needed

When influenza is circulating, laboratory confirmation of influenza is not needed before initiating antiviral treatment, as waiting until influenza is confirmed will delay initiation of therapy. Current clinical guidelines recommend that during influenza season, influenza antiviral therapy (e.g., oseltamivir) be started empirically for patients with:

Moderate, progressive, severe or complicated influenza, such as individuals who are hospitalized with influenza-like illness; OR
Compatible symptoms who are at high risk of influenza complications/severe illness.

Since these patients may require concurrent administration of empiric treatment for COVID-19 they may benefit from testing in order to guide the course of treatment.

For more information, please see Additional Respiratory Virus Testing Services at PHO’s laboratory below.

To understand which seasonal respiratory pathogens are circulating and to assist with influenza antiviral treatment decisions, healthcare providers are reminded to regularly review PHO’s Ontario Respiratory Virus Tool. The tool is updated weekly on Fridays and provides an overview of influenza and other respiratory pathogens circulating in the province. Data on influenza positivity is also presented at the local public health unit level to provide jurisdiction-specific information.

Interpretation

The following table provides possible test results with associated interpretations for FLUVID and MRVP:

Result	Interpretation	Comments
Negative	Virus not detected	Target virus not detected by real-time PCR
Positive	Virus detected	Target virus detected by real-time PCR
Indeterminate	Virus Indeterminate	An indeterminate PCR test result may be due to a low level of target genetic material in the specimen, inadequate specimen content, or a non-specific signal. Please resubmit another specimen for testing if clinically indicated.
Invalid	Invalid	Test results are invalid due to the failed amplification of the extraction control. Amplification failure may be due to inadequate specimen content, extraction failure, or PCR inhibition. Please resubmit another specimen for testing if clinically indicated.

Reporting

Results are reported to the ordering physician or healthcare provider as indicated on the requisition.

Specimens that are positive for SARS-CoV-2 and/or influenza, and all results from outbreaks, are reported to the Medical Officer of Health as per Health Protection and Promotion Act.

References

Fred Y Aoki, Upton D Allen, Samira Mubareka, Jesse Papenburg, H Grant Stiver, and Gerald A Evans. Use of antiviral drugs for seasonal influenza: Foundation document for practitioners—Update 2019. JAMMI. 2019 4:2, 60-82. Available at: jammi.utpjournals.press/doi/10.3138/jammi.2019.02.08
AMMI Canada Guidelines. Available at: ammi.ca/?ID=122
Antiviral Medications for Influenza: Information for Healthcare Providers. Available at: publichealthontario.ca/-/media/documents/q/2019/qa-antiviral-medicationinfluenza.pdf?la=en
2020–2021 AMMI Canada guidance on the use of antiviral drugs for influenza in the setting of co-circulation of seasonal influenza and SARS-CoV-2 viruses in Canada. Available at: https://doi.org/10.3138/jammi-2020-11-02

Data and Analysis

Rapport interactif

Cet outil offre des renseignements épidémiologiques complets sur l’activité des virus respiratoires en Ontario, y compris ceux de la COVID-19 et de la grippe. Explorez les tendances des cas,le dépistage en laboratoire, les éclosions ainsi que les résultats liés à la COVID-19 et la vaccination.

Additional Respiratory Virus Testing Services at PHO’s Laboratory

Influenza Antiviral Susceptibility Testing
Currently circulating influenza A subtypes (H1N1pdm09 and H3N2) are universally amantadine resistant. Almost all currently circulating influenza A and B viruses are oseltamivir susceptible; however, resistance has been documented on rare occasions.

Routine susceptibility testing is not required for clinical care; however, a proportion of influenza-positive samples will be forwarded to the National Microbiology Laboratory (NML) for strain typing and antiviral susceptibility testing. Oseltamvir resistance PCR screen is available if meeting the below criteria.

Recommended indications for antiviral susceptibility testing in Ontario include:
- Influenza developing during or soon after completion of influenza antiviral prophylaxis (e.g., oseltamivir).
- Severely ill patients such as those admitted to an ICU with laboratory-confirmed influenza not responding to influenza antiviral therapy.
- Fatalities in patients with laboratory-confirmed influenza being treated with influenza antiviral therapy.
- Persistent influenza viral shedding, defined as a repeat PCR test positive after seven days or more of treatment. Repeat PCR testing could be undertaken for patients who are not responding to antiviral therapy. Immunocompromised patients are at greater risk for more severe disease, persistent viral shedding and development of antiviral resistance.
- Positive test for influenza A in a traveler returning from an area where resistance is endemic.

To request influenza susceptibility testing for a patient who meets any of the above criteria, please document the request on the laboratory requisition along with any relevant information. To make a request on a sample already submitted to PHO, please contact our Customer Service Centre at 1-877- 604-4567 or 416-235-6556 or your local PHO laboratory.

Avian Influenza Viruses
PHO’s laboratory conducts testing for avian influenza (e.g., H5, H7, and N9) as required based on the information provided on the test requisition. Samples indicating travel to affected areas or with exposure to known cases of avian influenza, will be tested by real-time influenza A PCR (US CDC protocol) and if positive will be tested first for seasonal subtypes (H3N2, H1N1pdm09). If no seasonal subtype is detected, further testing for avian influenza subtypes will be conducted.

If you suspect avian Influenza, contact your local health unit and the PHO laboratory Customer Service Centre at 1-877-604-4567 prior to submitting specimens. Refer to the Avian Influenza Test Information Sheet.

Variant (swine origin) Influenza Viruses in Humans
Testing is available on request for variant influenza viruses, such as H3N2v, in persons who develop acute respiratory illness following direct contact with swine or their environment. Only limited human-to-human transmission has been documented.

NOTE: Samples that do not subtype for seasonal influenza will be tested for a panel of avian and/or swine influenza viruses.

Testing for Middle East Respiratory Syndrome Coronavirus (MERS-CoV)
MERS-CoV is a coronavirus that emerged in the Middle East in 2012. Patients with relevant clinical presentation and epidemiological risk factors for MERS-CoV infection, such as travel to certain countries in the Middle East or contact with a confirmed or probable MERS-CoV case, should be considered for MERS-CoV testing. For further information, please see the MERS-CoV Test Information Sheet.

Note: MRVP detects seasonal human coronavirus but does not detect MERS-CoV.

Testing for Enterovirus (including EV-D68 and other serotypes)
EV-D68 exhibits biennial circulation pattern with peaks during late-summer and autumn of even-numbered years.
For information about non-polio enterovirus, including D68, see PHO’s Infectious Disease page for Enterovirus D68.
For information on specimen collection and testing for enterovirus, see the Enterovirus Test Information Sheet.

Sentinel Practitioner Surveillance Network (SPSN)
For general information on Ontario’s SPSN and information for practitioners interested in contributing to the network, see Sentinel Practitioner Surveillance Network (SPSN) — Influenza Vaccine Effectiveness Program.

The SPSN performs molecular respiratory viral testing on patients with Acute Respiratory Infection (ARI) or influenza-like-illness (ILI) visiting community-based sentinel health care providers across Ontario. Other provinces also participate in the SPSN. SPSN sentinel heath care providers in Ontario will be able to offer influenza, other seasonal respiratory virus (by MRVP) and COVID-19 testing to patients meeting SPSN testing criteria, which includes outpatients who would not normally be eligible for influenza and other seasonal respiratory virus testing.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 01 mai 2025.

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Mis à jour le 1 avr. 2025