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) Screening and Genomic Sequencing for Surveillance.
- 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.
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 |
|
Preferred |
Preferred |
|
|
Preferred |
Acceptable |
|
| Preferred | Not Appropriate | |
|
Preferred |
Not Appropriate |
|
|
Saliva – neat – funnel6,7,8 |
Preferred |
Not appropriate |
|
Saliva: mouth rinse – “swish and gargle” – funnel6,7,8 |
Preferred |
Not appropriate |
|
Anterior Nares/Nasal/Nostril Swab (both sides) |
Acceptable |
Not Appropriate |
|
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 |
1 Refer to The Use of Alternate Sample Collection Methods for COVID-19 PCR Testing document for further information on alternate specimen types.
2 See 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 Supplychain.inquiries@ontario.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 |
Upper respiratory tract: Intended specimen Type: Nasopharyngeal swab (NPS) Possible alternative Specimen Types: Combined Throat and Both Nostrils Anterior Nares / Nasal / Nostrils Swab (both sides) Combined Oral (Buccal) and Deep Nasal Swab |
3 ml |
||
COVID-19 PCR |
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 |
Cerebrospinal fluid (CSF), other non-respiratory sterile fluid or tissue |
1.0 ml |
Sterile container |
Storage and 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
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.
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
OR
- 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.
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.
References
LAB-SD-139, New Test Requisition and Approval Process for COVID-19 Testing at Public Health Ontario
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