Respiratory Viruses (including influenza)

Testing Indications

Public Health Ontario (PHO) Laboratory utilizes a testing algorithm for influenza and other respiratory viruses.

On July 26, 2021, PHO Laboratory implemented changes to eligibility of multiplex respiratory virus PCR (MRVP) testing for children <18 years old seen in the Emergency Department, hospitalized patients, outbreak-associated patients, and patients in institutions not in outbreak with acute respiratory illness (ARI).

Previously, MRVP testing was routinely available for persons tested in ICU/CCU and remote communities only. Starting July 26, 2021, PHO Laboratory has made the following changes to MRVP testing:

  1. To support enhanced respiratory virus surveillance, MRVP testing will be available for symptomatic children (<18 years) seen in the Emergency Department (ED). This testing, which is generally not required for clinical purposes, will be re-evaluated in fall/winter 2021.
  2. MRVP testing will be available for all symptomatic hospitalized patients (ward and ICU/CCU).
  3. Specimens from the first four symptomatic patients in an outbreak that request respiratory virus testing will be tested by MRVP.
  4. Symptomatic patients tested in institutional settings (non-outbreak) will be eligible for MRVP testing when ordered on the PHO Laboratory requisition.

MRVP testing requests for patients with acute respiratory illness (ARI) in the settings described above should be clearly indicated on the requisition by selecting “Respiratory Viruses” or “COVID-19 Virus AND Respiratory Viruses” as appropriate in section 5 – “Test(s) Requested”. Only mark one of the three test request options. In addition, the patient’s setting and symptoms should be indicated on the requisition.

See Table footnote 5 in Test Methods for MRVP targets.

For information on COVID-19 testing see the COVID-19 Test Information Sheet (TIS)

Specimen Collection and Handling

Specimen Requirements

Test Requested Required Requisition(s) Specimen Type Minimum Volume Collection Kit

Respiratory Virus (Molecular)

Upper respiratory tract: Nasopharyngeal swab (NPS)

Nasopharyngeal swab in universal transport media (UTM)

Virus-Respiratory Collection Kit: order# 390082

Respiratory Virus (Molecular)

Deep Nasal swab

Deep Nasal swab in media provided with the collection kit

See table of acceptable alternative kits

Respiratory Virus (Molecular)

Viral throat swab (see Submission and Collection Notes below)

Swab in universal transport media (UTM)

Virus Culture Kit order#: 390081

Respiratory Virus (Molecular)

Lower respiratory tract (when possible): sputum, BAL, bronch wash, pleural fluid, lung tissue, tracheal aspirate (see Submission and Collection Notes below)

1.0 ml

Sterile container; Tuberculosis Kit order#: 390042

Submission and Collection Notes

1

To request respiratory virus testing only, check the “Respiratory Viruses” box in Section 5 of the COVID-19 Test Requisition (the preferred requisition during the COVID-19 pandemic). To request COVID-19 AND respiratory virus testing, check the “COVID-19 Virus AND Respiratory Virus” box in Section 5. Only select one of the three boxes.  

2

NPS is the most sensitive specimen type for respiratory virus testing. However, PHO Laboratory has done a limited evaluation on several COVID-19 specimen collection kits. 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 Laboratory and will not be accepted for testing respiratory viruses other than SARS-CoV-2.

3

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

  • Patient setting
  • Patient Date of Birth
  • Symptoms (indicate all symptoms)
  • Onset date
4

Patient setting (including outbreak or investigation number, if applicable) must be provided to help triage specimens. If patient setting is not provided, the specimen will only be tested for COVID-19.

5

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

6

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

CAUTION: “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 the rapid test and will be sent directly for MRVP or FLUVID testing.

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.

Preparation Prior to Transport

Label the specimen container with the patient’s full name, date of collection and one other unique identifier such as the patient’s date of birth or Health Card Number. See Criteria for Acceptance of Patient Specimens.

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 viability of the virus, specimens should be stored at 2-8°C following collection and transported to PHO Laboratory 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.

Special Instructions

Complete all fields of the COVID-19 Test Requisition (the preferred PHO Laboratory Requisition for respiratory virus testing during the COVID-19 pandemic), include the requesting healthcare provider's CPSO#, complete mailing address, telephone, and fax number, and the patient’s full name and address, date of birth, Health Card Number (must match the specimen label). Failure to do so may result in rejection or testing delay.

Test(s) requested must be clearly indicated in Section 5 of the requisition. Select ONE of the three options.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

The TAT for the Direct Antigen Test  for Influenza (Influenza rapid) is within 1 day after receipt at any PHO Laboratory site.

The TAT for seasonal respiratory virus testing is up to 4 days after receipt by PHO Laboratory.

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.

Test Methods

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

Respiratory virus testing is available according to the following tables:

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

(See Table 1b for testing of asymptomatic patients.)

Patient Setting1

Testing Available By Request

Hospitalized (all inpatients)2

 

 

SARS-CoV-2 and MRVP4,5,6
OR
FLUVID3,4 followed by MRVP4,5,6

(Both combinations will provide testing for the same viruses.)

Remote communities

SARS-CoV-2 and MRVP4,5,6
OR
FLUVID3,4 followed by MRVP

(Both combinations will 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] and school/daycare outbreaks.)

Up to 4 outbreak specimens7:

Influenza rapid testing8 (will be done if PCR testing is delayed >24 hours )

SARS-CoV-2 and MRVP4,5,6
OR
FLUVID3,4 followed by MRVP4,5,6

Additional specimens will be tested for SARS-CoV-2 only.7

Institutions (non-outbreak) (e.g. long-term care homes, correctional facilities, congregate living settings)11

SARS-CoV-2 and MRVP4,5,6
OR
FLUVID3,4 followed by MRVP4,5,6

Emergency Department; paediatric patients (<18 years old)12

 

SARS-CoV-2 and MRVP4,5,6
OR
FLUVID3,4 followed by MRVP4,5,6

Emergency Department; adult patients

SARS-CoV-29

Ambulatory/outpatient settings, assessment centres, including ambulatory influenza high risk patients

SARS-CoV-29

Not specified on requisition

SARS-CoV-21,9

 

Table 1b: Eligibility criteria for respiratory virus testing available at PHO Laboratory for asymptomatic patients

Patient Setting

Testing

All patient settings

Only SARS-CoV-2 testing will be performed on asymptomatic patients, regardless of patient setting

 

Table footnotes:

  1. The specific test being requested AND patient setting must also appear on the requisition to help with appropriate test assignment and triaging of specimens. If patient setting is not provided, the specimen will only be tested for SARS-CoV-2. For outbreaks or investigations, the requisition must include the assigned outbreak or investigation number.
  2. All ICU (Intensive Care Unit)/ CCU (Critical Care Unit) and ward inpatients.
  3. FLUVID detects: influenza A, influenza B, SARS-CoV-2 (COVID-19), and respiratory syncytial virus (RSV A/B). This assay may be used as an initial test prior to MRVP to provide earlier results during influenza and RSV seasons.
  4. Specimens first received at Kingston and Thunder Bay PHO Laboratory sites for MRVP testing may first undergo FLUVID testing (see 3 above), and will be forwarded to one of the MRVP testing sites (Toronto, London, Ottawa and Timmins) for MRVP testing. Specimens received at Hamilton site for MRVP will be tested for SARS-CoV-2 in Hamilton and forwarded to London or Toronto for further testing. Specimens received at Orillia, Peterborough or Sudbury sites will be forwarded to another PHO Laboratory site for molecular (PCR) testing.
  5. 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.
  6. A separate cytomegalovirus (CMV) PCR is available upon request (indicate on the requisition) for bronchoalveolar lavage (BAL)/bronchial wash/pleural fluid.
  7. A limit of four outbreak specimens are routinely accepted for seasonal respiratory virus testing if ordered on the PHO Laboratory requisition. For requests for additional testing in outbreak settings, contact PHO Laboratory’s Customer Service Centre. Respiratory outbreak specimens should be submitted following the Respiratory Outbreak Testing Prioritization Protocol
  8. Caution: influenza rapid testing can only be performed if specimen is received in suitable media. See Submission and Collection Notes above.
  9. 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 Laboratory.
  10. To assist with patient management when respiratory virus testing is not available, healthcare providers are encouraged to refer to PHO’s respiratory surveillance reports for information on respiratory pathogen activity in Ontario.
  11. Schools and daycare centres are not considered institutions for the purpose of sporadic MRVP testing.
  12. 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 during the summer and fall 2021. This will be re-evaluated in fall/winter 2021.

Table 2: Respiratory virus testing conducted at PHO Laboratory sites

PHO Laboratory Sites

Testing conducted

Management of specimens

Toronto, London, Ottawa, Timmins

Rapid Flu, SARS-CoV-2, FLUVID, MRVP

Specimens will be tested at receiving site

Hamilton

Rapid Flu, SARS-CoV-2

Specimens transferred to Toronto or London, if FLUVID or MRVP needed

Kingston, Thunder Bay

Rapid Flu, SARS-CoV-2, FLUVID

Specimens transferred to Toronto, London, Ottawa or Timmins if MRVP needed

Orillia, Peterborough, Sault Ste. Marie, Sudbury

Rapid Flu, no molecular testing

Specimens transferred to one of the above laboratory sites

 

When influenza is circulating, laboratory confirmation of influenza is not needed before initiating 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 or zanamivir) 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 disease.

For more information, please see the references provided 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 Pathogen Bulletin (ORPB). The ORPB is updated weekly 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.

 

Additional Respiratory Virus Testing Services at PHO Laboratory

  1. 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. Conversely, pre-pandemic seasonal influenza A (H1N1) was known to be amantadine susceptible, and almost universally oseltamivir resistant, but it has not been detected in Ontario since mid-2009.

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. Limited susceptibility testing is also available at PHO.

Recommended indications for antiviral susceptibility testing in Ontario include:

  • Influenza developing during or soon after completion of influenza antiviral prophylaxis (e.g., oseltamivir or zanamivir).
  • 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 traveller 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.

  1. Avian Influenza Viruses

PHO Laboratory conducts testing for avian influenza (e.g., H5N1, H5N2, and H7N9) 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 PHO Laboratory’s Customer Service Centre at 1-877-604-4567 prior to submitting specimens. Refer to the Avian Influenza Test Information Sheet.

In April 2013, the World Health Organization (WHO) confirmed emergence of new avian influenza A (H7N9) virus in China. For further information, including information on laboratory testing, see the Ontario Ministry of Health’s Guidance for Health Care Workers and Health Sector Employers on Asian lineage Avian Influenza A(H7N9).

Influenza A (H5N1) infections in humans have been infrequently reported since 2003 with cases occurring in Asia, and later in Africa, Europe, and the Middle East, mostly due to exposure to infected birds or their environment. For further information see the Ministry of Health’s H5N1 Flu Virus Fact Sheet.

  1. 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.

  1. 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.

  1. Testing for Enterovirus (including EV-D68 and other serotypes)

EV-D68 circulated in Ontario in the summer and fall of 2014, and to a lesser extent in 2016. More recently several cases of EV-D68 were detected at PHO Laboratory up to November 2018.
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.

References

  1. 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
  2. AMMI Canada Guidelines. Available at: ammi.ca/?ID=122
  3. Antiviral Medications for Influenza: Information for Healthcare Providers. Available at: publichealthontario.ca/-/media/documents/q/2019/qa-antiviral-medicationinfluenza.pdf?la=en
  4. 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

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Data and Analysis

Additional Information

For Outbreaks, contact your local Health Unit

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Updated 2 Oct 2021