Coronavirus Disease 2019 (COVID-19) – PCR

This document provides testing guidelines for the novel coronavirus disease 2019 (COVID-19, formerly known as 2019-nCoV) associated with an outbreak of respiratory illness that originated in Wuhan, Hubei Province, China in late 2019. The causative agent for COVID-19 disease is SARS-CoV-2 virus. For the purpose of clear communication, PHO uses the term COVID-19 to refer to both the virus and the disease.

Who to test:

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

Note: 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. Please note that given an incubation period of up to 14 days for COVID-19 disease after exposure, a negative PCR test result in an asymptomatic person should not be used to rule out disease. If the clinician would like to further discuss the role for testing, the PHO Microbiologists on-call 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.

To facilitate expeditious processing of specimens and avoid unnecessary delays, please ensure all mandatory information is included on the specific PHO COVID-19 Test Requisition.

As of March 2, 2020, the respiratory virus multiplex PCR (MRVP) will no longer be performed on all specimens submitted for COVID-19 testing. MRVP will 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 the Respiratory Viruses Test Information Sheet for more details and acceptance criteria.

Specimen Collection and Handling

Due to global shortages, the following chart provides information on alternative collection kits for procurement that are acceptable for COVID-19 testing.

At this time there are no dedicated neonatal nasopharyngeal swabs available in Ontario. Current advice is to 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 as alternative collection sites.

If you are ordering collection kits from PHOL.Warehouse@oahpp.ca using the Requisition for Specimen Containers and Supplies Form any of these kits may be used to fulfill orders (see submission note #3 below).

Manufacturer/
Vendor Catalogue

Description

Recommended Specimen Type Special Instructions* Suitable for
(R=Flu Rapid**; C=COVID-19; M=Multiplex)

BD
B220528

3mL Vial, one sterile Nylon flocked tip swab, scored plastic shaft

Deep Nasal or Throat swab Steps for NPS collection C,M

BD
B220531

3mL Vial, one sterile mini-tip swab, scored plastic shaft

Nasopharyngeal swab (NPS)   C,M
Beaver Biomedical Engineering
43903
Sample Collection Kit – 3mL of pink VTM with Specimen Collection swab (80mm breakpoint) Nasopharyngeal swab (NPS) Steps for NPS collection R,C,M
Beaver swab paired with Trinity Biotech Medium/B1029-90D Beaver swab (80mm breakpoint) Bartels media (tube cap may vary in colour) Nasopharyngeal swab (NPS) Steps for NPS collection R,C,M
Copan/220252 paired with PBS media Copan FLOQ Swab (100mm breakpoint) PBS Media (tube cap may vary in colour and tube vial may vary in shape) Nasopharyngeal swab (NPS) Steps for NPS collection R,C,M
Copan/220252 paired with Roche/Cobas PCR media Copan FLOQ Swab (100mm breakpoint) Roche PCR media Nasopharyngeal swab (NPS) Steps for NPS collection C,M

Copan
CA302MH

3mL UTM standard flock

Deep Nasal or Throat swab Steps for deep nasal collection R,C,M

Copan
CA305MH

3mL UTM Pernasal flock

Nasopharyngeal swab (NPS)   R,C,M

Copan
CA56750CS01

Midturbinate - pediatrics swabs

Deep Nasal or Throat swab

Steps for deep nasal collection

Use only to collect from pediatric patients

R,C,M
Deaou kits Virus Sampling Tube – 3mL VTM with Disposable Sampling swab (80mm breakpoint) Nasopharyngeal swab (NPS) Steps for NPS collection C,M

ESBE
EZPro
ESB-VA50005NP

EZPro Nasopharyngeal Swab Kit with Clear Transport Medium

Nasopharyngeal swab (NPS) C,M
GDL Korea 2mL of pink UTM with Nylon Flocked swab (95mm breakpoint) Nasopharyngeal swab (NPS) Steps for NPS collection R,C,M

Hologic
PRD-03546

Multitest Swabs

Deep Nasal or Throat swab Steps for NPS collection C,M

Hologic
#301041

Unisex Test Swabs

Deep Nasal or Throat swab

Steps for NPS collection

Please note that this kit contains a cleaning swab that is not suitable for testing.

C,M
Miraclean kits Disposable Sampling Kit – 2mL of pink VTM with Disposable Sampling swab (95mm breakpoint) Nasopharyngeal swab (NPS) Steps for NPS collection R,C,M
Miraclean swab paired with McMaster Molecular Medium Miraclean swab (95mm breakpoint) McMaster Molecular Medium Nasopharyngeal swab (NPS) Steps for NPS collection C,M
Miraclean swab paired with Roche/Cobas PCR media Miraclean swab (95mm breakpoint) Roche PCR media Nasopharyngeal swab (NPS) Steps for NPS collection C,M
Miraclean swab paired with Trinity Biotech Medium/B1029-90D Miraclean swab (95mm breakpoint) Bartels media (tube cap may vary in colour) Nasopharyngeal swab (NPS) Steps for NPS collection R,C,M

Norgen
i-Clean

Narrow- tipped flocked swab with medical-grade nylon microfibers, 80mm breakpoint, three-step shaft thickness

Nasopharyngeal swab (NPS)

 

C,M

Norgen
i-Clean/CY-93050

Specimen collection swab with thick shaft

Deep Nasal or Throat swab

 

Steps for deep nasal collection

Please note this swab may look similar to the NPS Norgen i-Clean, but the CY-93050 does not have a narrow tip or three-step shaft.

C,M

Norgen i-Clean/CY-96000 paired with PBS Media

i-Clean swab (narrow- tipped flocked swab, 80mm breakpoint, three-step shaft thickness)

PBS Media (tube cap may vary in colour and tube vial may vary in shape)

Nasopharyngeal swab (NPS)

 

R,C,M

Norgen i-Clean/CY-96000 paired with Roche/Cobas PCR media

i-Clean swab (narrow-tipped flocked swab, 80mm breakpoint, three-step shaft thickness)

Roche PCR media

Nasopharyngeal swab (NPS   C,M

Oxoid
OXRE12568

M6 Kit with Traditional-Tipped Flocked Swab

Deep Nasal or Throat swab

Steps for deep nasal collection

R,C,M

Oxoid
OXRE12569

M6 Kit with Micro-Tipped Flocked Swab

Nasopharyngeal swab (NPS)   R,C,M

Puritan/25-3317-H paired with PBS Media

Puritan swab (105mm breakpoint)

PBS Media (tube cap may vary in colour and the tube vial may vary in shape)

Nasopharyngeal swab (NPS)

 

R,C,M

Puritan/25-3317-H paired with Roche/Cobas PCR media

Puritan swab (105mm breakpoint)

Roche PCR media  

Nasopharyngeal swab (NPS)

 

C,M

Puritan/25-3317-H paired with Remel M4RT Puritan swab (105mm breakpoint) Remel M4RT media Nasopharyngeal swab (NPS) Steps for NPS collection R,C,M
Roche/CHS
85-1051
Roche Woven tip Swab (70mm breakpoint) with Cobas PCR media Deep Nasal or Throat swab

Steps for deep nasal collection

Steps for throat collection

C,M
Roche/CHS
85-1052
Roche Flocked tip Swab (70mm breakpoint) with Cobas PCR media Deep Nasal or Throat swab

Steps for deep nasal collection

Steps for throat collection

C,M

Roche
07958021190

KIT COBAS PCR MEDIA DUAL SWB

Deep Nasal or Throat swab

Steps for deep nasal collection

Please note that this kit may contain 2 swabs, either can be used for specimen collection.

C,M
Roche
07958021190
KIT COBAS PCR MEDIA UNI SWAB specimen
Deep Nasal or Throat swab

Steps for deep nasal collection

C,M
Sensor Health swab paired with Trinity Biotech Medium/B1029-90D Sterile “Sampling Swab” (80mm breakpoint) Bartels media (tube cap may vary in colour) Nasopharyngeal swab (NPS) Steps for NPS collection R,C,M

Sensor Health swab paired with Roche/Cobas PCR media

Sterile “Sampling Swab” (80mm breakpoint)

Roche PCR media  

Nasopharyngeal swab (NPS)

C,M

Sensor Health kit paired with PBS Media

Unscored swab with scissors   

PBS Media (tube cap may vary in colour and the tube vial may vary in shape)

Deep Nasal or Throat Swab

 

Steps for cutting swab

Steps for deep nasal collection

Steps for throat collection

R,C,M

Trinity Biotech Bartels Flextrans Transport medium
(B1029-90D) paired with i-Clean swab

Bartels media (tube cap may vary in colour)

i-Clean swab (narrow-tipped flocked swab, 80mm breakpoint, three-step shaft thickness)

Nasopharyngeal swab (NPS) R,C,M
VWR/ Starplex
CA66410-030
Coll, Multitrans Male STR  Deep Nasal or Throat

Steps for deep nasal collection

R,C,M 

Yocon- Virus sampling kit with VTM

Flocked swab (80mm breakpoint) with pink viral transport media

Nasopharyngeal swab (NPS)

 

R,C,M


*
For instructions on upper respiratory specimen collection, please follow the US CDC’s information for health professionals influenza specimen collection poster.

**Flu Rapid refers to the immunochromatographic rapid influenza test used for institutional outbreak testing; for more information refer to the Respiratory Viruses Test Information Sheet (including influenza) for Flu outbreak testing algorithm.

Specimen Requirements

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

COVID-19

Upper respiratory tract: Nasopharyngeal swab (NPS)

Nasopharyngeal swab in 3 ml universal transport media (UTM)

Virus Respiratory Kit order # 390082

COVID-19

Deep Nasal swab

Deep Nasal swab in 3 ml media provided with the collection kit

See previous table of acceptable alternative kits

COVID-19

Viral throat swab (see Submission and Collection Notes below)

Swab in 3 ml universal transport media (UTM)

Virus Culture Kit order #390081

COVID-19

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

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), deep nasal swab OR viral throat swab collected in universal transport medium (UTM) or alternative media as described above. NPS is the preferred specimen when swabs are available, followed by a deep nasal swab.

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, 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.
2

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.

 
3

Alternative specimen collection kits

PHO Laboratory has done a limited evaluation on the alternative specimen collection kits listed above. These can be used for collection of upper respiratory specimens as indicated in the table above, for COVID-19 testing. The same specimen can be used for seasonal respiratory virus testing (MRVP) provided acceptance criteria are met (see above).

PHO Laboratory will accept other swab types and other liquid transport media (except gel or solid media) for COVID-19 testing; however, if the submitted swab and/or media type were not validated at PHO Laboratory for COVID-19 testing, the results will be reported with a disclaimer. Further information on alternative collection kits is available here: FAQs on Diagnostic Testing for SARS-CoV-2.

 
4

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
  • 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)

Preparation Prior to Transport

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 in accordance with the Transportation of Dangerous Goods Regulations.

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

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Testing for COVID-19 is performed at PHO Laboratory as required. NOTE: Turnaround time will vary according to geographical location and proximity to a PHO Laboratory location that performs COVID-19 testing.

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

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. Current PHO Laboratory testing locations include Toronto, Hamilton, Kingston, Timmins and Ottawa. 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
  • URGENT samples must be placed in a clearly marked package indicating ‘URGENT’ and handled in accordance with the Canadian Biosafety Standards and shipped in accordance with the Transportation of Dangerous Goods Regulations.
  • Failure to ship separately will delay testing, primarily due to delays in transportation, and will be processed as a routine sample.

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. Commercial assays are also in use at PHO Laboratory, and targets vary across the assays as outlined below.
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

Abbott

N gene, RdRp gene

E – envelope; Orf1a/b – open reading frame 1a/b; RdRp – RNA dependent RNA polymerase; N – Nucleocapsid
*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, the more sensitive of the two PCR targets.
    • 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 PHOL 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 controls. 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 alternative testing assay at PHO Labortatory.

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.

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.

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. 

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:

i. Mycoplasma pneumoniae/Chlamydia pneumoniae duplex PCR:

  • Note: routine Mycoplasma/Chlamydia testing of respiratory specimens has been temporarilty suspended due to the pandemic. If testing is still required on a hospitalized patient, please contact PHO Laboratory Customer Service. This testing can be performed on the same respiratory specimen submitted for COVID-19 testing.

ii. Legionella testing:

NB. 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.

Data and Analysis

Mis à jour le 23 juill. 2020