Coronavirus Disease 2019 (COVID-19) – PCR

This page provides testing guidelines for COVID-19. The causative agent for COVID-19 disease is SARS-CoV-2 virus.

Updates

The following test information has been revised. Please refer to the section or link for full details.

Updates to “Specimen Types for COVID-19 testing by Patient Characteristic” including the addition of Combined Oral (Buccal) and Deep Nasal specimens, and updates to Saliva as a specimen type
For information on SARS-CoV-2 Variant of Concern (VoC) surveillance, see the SARS-CoV-2 (COVID-19 Virus) Variant of Concern (VoC) Surveillance Test Information Sheet.
Saliva was added as an acceptable sample for COVID-19 PCR testing. Only a limited number of non-PHO laboratories in Ontario are accepting saliva for COVID-19 PCR testing.
Focus On COVID-19: Cycle Threshold Values, 16 September 2020
Evidence Brief: Use of Alternative Sample Collection Methods for COVID-19 PCR Testing, 15 September 2020
Added links to the Respiratory Outbreak Testing Prioritization Protocol, 11 November 2020

Who to Test

Please refer to the testing guidelines from the Ministry of Health.

Given an incubation period of up to 14 days for COVID-19 disease after exposure, a negative PCR test result in an asymptomatic person cannot be used to rule out exposure and subsequent disease. PHO is not currently recommending routine testing of asymptomatic persons for COVID-19 outside of those recommended in Ministry guidance, or as directed by the public health unit for public health investigation.
If a healthcare provider would like to further discuss the role for testing, the on-call PHO Microbiologists are available and can be contacted through the PHO Laboratory's Customer Service Centre at 416-235-6556 / 1-877-604-4567 or the After-Hours Emergency Duty Officer at 416-605-3113.

*Uncontrolled print copy. Valid only on day of print: 25 May 2021.

Specimen Collection and Handling

Specimen Types for COVID-19 testing by Patient Characteristic

	Patient Characteristics
Specimen Type¹	Non-hospitalized patients with respiratory symptoms or asymptomatic persons meeting criteria outlined by the Ministry of Health	Hospitalized patients with respiratory symptoms
Nasopharyngeal (NP) swab²	Preferred	Preferred
Combined swab of Throat and both Nares³	Preferred	Acceptable
Combined Oral (Buccal) and Deep Nasal Swab⁴	Preferred	Not Appropriate
Deep Nasal swab⁵	Preferred	Not Appropriate
Saliva – neat – funnel^6,7,8 Saliva – neat – straw^6,7,8	Preferred	Not appropriate
Saliva: mouth rinse – “swish and gargle” – funnel^6,7,8 Saliva: mouth rinse – “swish and gargle” – straw^6,7,8	Preferred	Not appropriate
Anterior Nares/Nasal/Nostril Swab (both sides)	Acceptable	Not Appropriate
Throat/Oropharyngeal swab	Acceptable	Not Appropriate
Lower respiratory tract (when possible): sputum, BAL, bronch wash, pleural fluid, lung tissue, tracheal aspirate	Acceptable	Preferred
Cerebrospinal fluid (CSF), other non-respiratory sterile fluid or tissue	Acceptable	Acceptable

¹Refer to The Use of Alternate Sample Collection Methods for COVID-19 PCR Testing document for further information on alternate specimen types.
²See Specimen Collection Kits and Instructions Table below for which kit is also suitable for Rapid Flu testing and seasonal respiratory multiplex testing.
³Combined Throat and both nares specimens approximate sensitivity of NPS [LeBlanc JJ et el. A combined oropharyngeal/nares swab is suitable alternative to nasopharyngeal swabs for detection of SARS-CoV-2. J Clin Virol. 2020;128: 10442. https://doi.org/10.1016/j.jcv.2020.104442]
⁴Combined Oral and both nares approximate sensitivity of NPS [Gertler M et al. Self-collected oral, nasal and saliva samples yield sensitivity comparable to professional-collected oro-nasopharyngeal swabs in SARS-CoV-2 diagnosis https://www.medrxiv.org/content/10.1101/2021.04.13.21255345v1]
⁵Canadian Public Health Laboratory Network Best Practices for COVID-19 V1.0 April 23, 2020
⁶In a meta-analysis and systematic review, saliva NAAT had a similar sensitivity and specificity to that of nasopharyngeal NAAT [Butler-Laporte G, et al. Comparison of Saliva and Nasopharyngeal Swab Nucleic Acid Amplification Testing for Detection of SARS-CoV-2: A Systematic Review and Meta-analysis. JAMA Intern Med. 2021;181(3):353–360. doi:10.1001/jamainternmed.2020.8876]
⁷Saliva sample should not be collected from symptomatic patients residing in institutional settings (e.g. retirement homes or long-term care facility) or hospitalized patients.
⁸Saliva samples are not suitable and cannot be accepted for testing for respiratory viruses (e.g. influenza virus, RSV) other than SARS-CoV-2.

COVID-19 PCR Collection Kits

There are a number of acceptable collection kits for COVID-19 PCR testing.

Effective September 10, 2020 – to obtain COVID-19 swab kits, please submit your order via the Ontario Health Digital Health Services page.

If you have any further inquiries about ordering COVID-19 PCR collection kits, please reach out to:

Toronto: Robert Burgess (Robert.Burgess@sunnybrook.ca) and Nancy Kraetschmer (nancy.kraetschmer@tc.lhins.on.ca)
North: covidsuppliesnorth@tbh.net
East: Paul McAuley (Paul.McAuley@3SO.ca)
West: HMMSCOVID19@hmms.on.ca
Central: OHCentralPPEinquiries@lhins.on.ca

Note: Nasopharyngeal Swabs for Neonates
At this time there are no dedicated neonatal nasopharyngeal swabs available in Ontario. Collect an NP swab when testing neonates for COVID-19. If the calibre of the swab that is available is too wide, it can be used for nasal, deep nasal or throat swab collection sites.

FAQ
Questions regarding swabs, kits and media as well as testing results and performance are answered in our FAQs.

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
COVID-19 PCR	Coronavirus Disease 2019 (COVID-19) Test Requisition	Upper respiratory tract: Intended specimen Type: Nasopharyngeal swab (NPS) Possible alternative Specimen Types: Combined Throat and Both Nostrils Deep Nasal swab Anterior Nares / Nasal / Nostrils Swab (both sides) Combined Oral (Buccal) and Deep Nasal Swab Throat / Oropharyngeal swab Saliva - neat – funnel Saliva – neat - straw Saliva: mouth rinse – “swish and gargle” – funnel Saliva: mouth rinse – “swish and gargle” – straw	3 ml	Virus Respiratory – Acceptable Kits
COVID-19 PCR	Coronavirus Disease 2019 (COVID-19) Test Requisition	Lower Respiratory Tract (when possible): sputum, BAL, bronch wash, pleural fluid, lung tissue, tracheal aspirate (see Submission and Collection Note #1 below)	1 ml	Sterile container
COVID-19	Coronavirus Disease 2019 (COVID-19) Test Requisition	Cerebrospinal fluid (CSF), other non-respiratory sterile fluid or tissue	1.0 ml	Sterile container

Submission and Collection Notes

Respiratory Tract Specimens

Patients not admitted to hospital (including those in ER)

A single upper respiratory tract specimen will be accepted for COVID-19 testing.

Upper respiratory tract specimens include: a nasopharyngeal swab (NPS), combined oropharyngeal/throat plus both anterior nares/nostrils, combined oral (buccal) and deep nasal swab, deep nasal swab, oropharyngeal/throat swab, or swab of both anterior nares, collected in universal transport medium (UTM) or other viral media as described above.
Upper respiratory tract specimens can also include saliva collected neat or as a saline mouth rinse “swish and gargle”. Please see all notes associated with this specimen type in the table “Preferred and Acceptable Specimen Types for COVID-19 testing by Patient Characteristic” above. Saliva sample should not be collected from symptomatic patients residing in institutional settings (e.g. retirement homes or long-term care facility) or hospitalized patients. Saliva samples are not suitable and cannot be accepted for testing for respiratory viruses (e.g. influenza virus, RSV) other than SARS-CoV-2 NOTE: only limited non-PHO laboratories are accepting saliva testing for COVID-19 PCR.

In-patients

For in-patients, PHO Laboratory will accept multiple specimen submissions. If done, this should be done from different collection sites:

Upper respiratory tract: submit NPS, combined swab of oropharyngeal/throat plus both anterior nares/nostris, or deep nasal swab. A viral throat swab collected in UTM may be submitted as an additional specimen, but is not required. PHO Laboratory will accept multiple swabs (e.g NP swab, throat swab) placed into one transport medium. This combined submission will be run and reported as a single test.
Lower respiratory tract specimens: submit when possible.
Sputum: collect if patient has a productive cough. Do not induce.

Use of laboratory testing for clearance of confirmed and probable COVID-19 cases from isolation

For each scenario below, cessation of isolation requires that the individual is afebrile and symptoms are improving.

For confirmed and probable cases at home: Laboratory testing is not required for discharge from isolation. These patients can be discharged from isolation and categorized as “resolved” 14 days following symptom onset.
For hospitalized patients: Isolate in hospital until two negative tests, obtained at least 24 hours apart. If discharged home within 14 days of symptom onset, follow advice for individuals at home.

For further information, please refer to the Ministry of Health COVID-19 Quick Reference Public Health Guidance on Testing and Clearance.

Respiratory Virus Multiplex PCR (MRVP)

As of March 2, 2020, MRVP is no longer performed on all specimens submitted for COVID-19 testing. MRVP is only be performed by request for patients that meet PHO Laboratory’s acceptance criteria for this testing (e.g. hospitalized, outbreaks, institutionalized). If patients meet testing criteria, MRVP can be ordered on the same swab submitted for COVID-19 testing. Refer to Respiratory Viruses Test Information for more details and acceptance criteria.

Complete all fields of the COVID-19 Test Requisition.

Test(s) requested and indication for testing (If confirmatory or clearance testing is requested, this must be indicated on the test requisition)
Travel history (if applicable)
Exposure history and details
Clinical information including symptom onset date or if asymptomatic
COVID-19 vaccination status
Specimen type and collection
Patient setting/population (If the patient belongs to a priority group, as per the Ministry of Health testing guidance, this must be indicated on the requisition)

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

Preparation Prior to Transport

For outbreak specimens follow the Respiratory Outbreak Testing Prioritization Protocol.

Place specimen in biohazard bag and seal. Specimens should be stored at 2-8°C following collection and shipped to PHO Laboratory on ice packs. If transport of specimen to testing laboratory will be delayed more than 72 hours, specimens should be frozen at -70°C or below and shipped on dry ice.

Package and ship primary clinical specimens to the local PHO Laboratory or directly to a PHO Laboratory conducting COVID-19 testing (see list of COVID testing laboratory sites, below) in accordance with the Transportation of Dangerous Goods Regulations. You may use these shipping labels for packages that are not marked with Transportation of Dangerous Goods signs.

Current PHO Laboratory testing locations include Toronto, Hamilton, Kingston, London, Ottawa Timmins and Thunder Bay. Refer to specimen acceptance hours and testing schedules at PHO Laboratory COVID-19 testing locations.

Labels for Packages Containing Test Samples | Étiquettes pour les emballages contenant des échantillons d’épreuve

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

COVID-19 testing is performed 7 days a week at PHO Laboratory. TAT: 60% of results are to be completed within 24 hours and 80% to be completed within 48 hours.

Turnaround time may vary according to geographical location and proximity to a PHO Laboratory location that performs COVID-19 testing.

Saliva or Mouth Rinse (“swish and gargle”) specimens may have turn around times that are longer than for NPS, nasal and throat swab specimens.

Testing for COVID-19 does not need to be sent as STAT specimens unless the result is needed to assist health-care providers in making swift patient care decisions in an urgent or emergency circumstance.

URGENT samples must be shipped separately from routine specimens to a PHO Laboratory location that performs COVID-19 testing. Failure to ship COVID specimens separately may delay testing, primarily due to delays in transportation. Specimen may inadvertently be processed as a routine sample.

URGENT samples must be placed in a package that has been clearly marked ‘URGENT’ and handled in accordance with the Canadian Biosafety Standards and shipped in accordance with the Transportation of Dangerous Goods Regulations.
PHO Laboratory COVID-19 testing locations include Toronto, Hamilton, Kingston, London, Ottawa, Timmins and Thunder Bay. Refer to specimen acceptance hours and testing schedules at PHO Laboratory COVID-19 testing locations.
For testing at PHO Laboratory Toronto, specimens must be sent to the shipping and receiving dock at 661 University Ave., Toronto, Ontario. For delivery instructions please see Directions to 661 University Shipping Dock for Clinical Samples.

Reporting

Final results of COVID-19 testing from PHO Laboratory are reported to the ordering health-care provider as indicated on the requisition.

As a disease of public health significance, all positive results will be reported to the local public health unit.

If specimens submitted to PHO Laboratory are forwarded to another laboratory for testing as part of Ontario’s response to the COVID-19 pandemic, the reporting of results to the submitter as well as any required health unit notifications will be performed by the testing laboratory.

Test Methods

Testing for COVID-19 is done by real-time PCR using protocols validated by PHO Laboratory and the NML. Several specimen collection kits have been extensively evaluated on a number of different extraction and PCR platforms at PHO. To meet increasing submissions for COVID-19 testing, PHO Laboratory has validated and instituted a variety of commercial assays which are validated for clinical testing. The assay used will be identified on the report.

Current Assays in Use at PHO Laboratory and Associated Gene Targets

Assay	Gene Targets
PHO Laboratory LDT	E gene*
Roche	Orf1a/b gene, E gene
Thermofisher Amplitude	Orf1a/b gene, S gene, N gene

E – Envelope; Orf1a/b – open reading frame 1a/b;; N – Nucleocapsid , S- Spike
*Specimens may also be tested using a laboratory developed RdRp gene target assay

Specimens tested using the in-house laboratory developed assay will be tested using the E gene real-time PCR assay, which is more sensitive than the RdRp gene target.

Specimens with a single target detected (regardless of assay used) will be reported as COVID-19 virus detected, which is sufficient for laboratory confirmation of COVID-19 infection.
Specimens with no gene target(s) detected in the assay used will be reported as COVID-19 virus not detected.

Specimens with a gene target result which is indeterminate* may be investigated further.

Additional testing will include real-time PCR on an alternate assay (see table above). Sanger sequencing for the COVID-19 virus RdRp gene may also be conducted.
Subsequent detection of COVID-19 virus by any single gene target with a validated real-time PCR assay, or alternatively by the RdRp gene sequencing assay, is sufficient for laboratory confirmation.

Laboratory confirmation of COVID-19 at PHO Laboratory consists of:

Detection of at least one genome target by real-time PCR

One or multiple targets is/are indeterminate* by real-time PCR AND detection of COVID-19 virus by sequencing.

PHO Laboratory Testing is considered INCONCLUSIVE if:

One or multiple targets is/are indeterminate* by real-time PCR.

AND

COVID-19 is not detected (or remains indeterminate) by testing on an alternative real-time PCR assay, or on Sanger sequencing of the RdRp gene.

*An indeterminate result on a real-time PCR assay is defined as a late amplification signal at a predetermined high cycle threshold (Ct) value range (Ct >38 in the PHO COVID-19 laboratory developed assay). This may be due to low viral target quantity in the clinical specimen approaching the limit of detection of the assay, or alternatively may represent nonspecific reactivity (false signal) in the specimen. When clinically relevant, indeterminate samples should be investigated further by testing for an alternate gene target using a validated real-time PCR or nucleic acid sequencing assay at the community, hospital or reference laboratory that is equally or more sensitive than the initial assay or method used.

Specimens will be sent to NML for further testing if required.

A test is reported as INVALID when:

Results are uninterpretable. This could be for various reasons, most commonly due to failed detection of assay control. Each invalid result is accompanied with a specific comment to explain the cause. When possible, specimens that are invalid are repeated on the same test system and/or retested on an alternate testing assay at PHO Laboratory.

Case Confirmation

Some hospital and community laboratories have implemented COVID-19 testing in-house and report final positive results, which is sufficient for case confirmation. Other hospital and community laboratories will report positives as preliminary positive during the early phases of implementation and will require confirmatory testing at PHO Laboratory. If confirmatory testing is requested, this should be indicated on the test requisition by the submitting hospital or community laboratory.

Details about the laboratory developed real-time PCR used at PHO Laboratory for COVID-19 testing are available at: Diagnostic detection of COVID-19 by real-time RT-PCR.

The following publication contains additional technical information on the RdRp gene PCR and sequencing assay which PHO Laboratory has adapted to be specific for COVID-19 virus detection: Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections.

Algorithm

PHO Laboratory Testing Algorithm for COVID-19 (as of March 16, 2020)

A. Testing for COVID-19

See Test Methods above.

B. Testing for Other Respiratory Viruses:

Testing for other respiratory viruses by multiplex respiratory virus PCR (MRVP) can be ordered if patients meet PHO Laboratory’s acceptance criteria for this testing (e.g. hospitalized, outbreaks, institutionalized). Refer to the Respiratory Viruses Test Information Sheet for more details and acceptance criteria.
*Cross-reaction with COVID-19 has not been detected based on in-house laboratory data and available sequence data.
Testing for avian influenza can be ordered if patients meet criteria for this testing. See the Avian Influenza Test Information Sheet for more details.

Note: Testing for other respiratory viruses and avian influenza must be ordered on the laboratory requisition if required and can be ordered on the same swab submitted for COVID-19 testing.

Additional Tests to be Considered

Testing for bacterial causes of community-acquired pneumonia:

Patients with pneumonia/parenchymal lung involvement should also be tested for bacterial causes of community acquired pneumonia (CAP). Recommended testing available at PHO Laboratory includes:

Mycoplasma pneumoniae/Chlamydia pneumoniae duplex PCR:
- This testing can be performed on the same respiratory specimen submitted for COVID-19 testing.
Legionella testing:
- Legionella PCR: can be ordered on the same lower respiratory tract specimens submitted for COVID-19 testing (e.g. sputum, BAL, bronchial wash, aspirates, lung tissue).
- Legionella urinary antigen testing (minimum urine volume 2 ml).

Note: If a person under investigation for COVID-19 is worsening or not improving, testing should be repeated, even if previous tests were positive for another pathogen.