Coronavirus Disease 2019 (COVID-19) – PCR

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 Test Information Sheet provides information on routine polymerase chain reaction (PCR) (molecular) testing for SARS-CoV-2, the causative agent of COVID-19. For other testing modalities, please refer to the following links:

Additional tests to be considered: Bacterial causes of community-acquired pneumonia:|
Patients with pneumonia/parenchymal lung involvement may also be tested for bacterial causes of community acquired pneumonia (CAP). Recommended testing available at PHO’s laboratory includes:

Updates

Effective October 1, 2024, the Ministry of Health has updated COVID-19 testing eligibility. For expanded criteria on testing eligibility for patients at higher risk of severe illness or outcomes from Influenza and COVID-19, visit PHO’s webpage.
Patient setting must be indicated on the requisition for samples to qualify for testing. Specimens submitted without patient setting will be rejected.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 15 janv. 2025.

Testing Indications

Who to test:
For test indications, please refer to the testing guidance documents from the Ontario Ministry of Health. As of October 1, 2024, asymptomatic patients are no longer eligible for COVID-19 testing and specimens from asymptomatic patients will be rejected for testing.

Acceptance/Rejection Criteria

Specimen Collection Options for COVID-19 PCR Testing by Patient Characteristic

Specimen Collection Method	General Recommendations	Approximate Sensitivity Range^a
Nasopharyngeal (NP) swabbing	Preferred option for all patients (hospitalized or non-hospitalized)^b	95-100% ^{1, 2, 3, 4, 5, 6, 8, 9}
Combined oral/oropharyngeal (buccal/throat) and nasal (midturbinate) swabbing	Other preferred option for non-hospitalized patients^b	85-95% ^{1, 2, 10, 13, 14}
Saliva (neat or saline rinse)	Other preferred option for non-hospitalized patients^b,d	85-95% ^{1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12}
Lower respiratory tract (when possible): sputum^c, BAL, bronchial wash, pleural fluid, lung tissue, and tracheal aspirate	Other preferred option for hospitalized patients	95-100% ¹
Nasal (midturbinate) swabbing	Acceptable option for non-hospitalized patients	80-85% ^{1, 3, 7, 8, 9, 10, 11, 12, 13, 14}
Throat swabbing	Acceptable option for non-hospitalized patients	80-85% ^{1, 13, 14}
Non-respiratory: cerebrospinal fluid (CSF), other non-respiratory sterile fluid or tissues	Acceptable option for hospitalized patients	Not defined^e,1

^a Sensitivity may vary depending on training, collection quality, disease timeline, disease severity, virus lineage, and many other factors, therefore ranges are provided as approximate reference only. Values are approximated.

^b NP swabbing usually provides the highest sensitivity, however it may be less tolerated for some patients and is a regulated procedure, therefore not all settings can perform it. Alternatively, the combined oral and nasal swabbing option and the saliva option both provide relatively high sensitivity in the early infection phase and are suitable for most outpatient settings where NP swabbing is challenging to operationalize. Patients at risk of developing severe disease may still benefit from the higher sensitivity of NP swabbing. Risk factors for severe disease are listed in the Ontario Ministry of Health testing guidance.

^c Only if patient has self-productive cough (do not induce).

^d Saliva may be less feasible to collect in some patient populations with reduced salivary production or inability to reliably salivate into the collection tube. All PHO locations accept saliva specimens for COVID-19 PCR testing. However, some non-PHO laboratories may not be able to accept this specimen type. Contact your local laboratory provider if you have questions regarding the availability of COVID-19 saliva testing. Testing of respiratory viruses (including influenza) other than SARS-CoV-2 is not available on saliva specimens. Settings requiring testing of both SARS-CoV-2 and other respiratory viruses should select other collection options.

^e Considering the limited evidence for COVID-19 testing on non-respiratory specimens, these specimen types should not be routinely ordered. Testing should be reserved for atypical situations strongly suggestive of COVID-19 infection outside of the respiratory tract, and results should be interpreted with caution as test performance has not been verified for these specimen types. In such situations, common etiologies causing infections outside of the respiratory tract should be considered.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
COVID-19 PCR	COVID-19 and Respiratory Virus Test Requisition	Upper respiratory tract: Nasopharyngeal (swab) NPS	2.0 ml (including collection media)	Virus-Respiratory Collection Kit: order #390082
COVID-19 PCR	COVID-19 and Respiratory Virus Test Requisition	Upper respiratory tract: combined oral and nasal swabbing, nasal swabbing, or throat swabbing	Swab in universal transport media (UTM)	Virus PCR Kit order#: 390081
COVID-19 PCR	COVID-19 and Respiratory Virus Test Requisition	Saliva (neat or swish and gargle)	2.0 to 3.0 mL	Sterile container as provided in collection kit
COVID-19 PCR	COVID-19 and Respiratory Virus Test Requisition	Lower respiratory tract (when possible): sputum, BAL, bronchial wash, pleural fluid, lung tissue, tracheal aspirate	1.0 ml (if applicable)	Sterile container
COVID-19	COVID-19 and Respiratory Virus Test Requisition	Non-respiratory: Cerebrospinal fluid (CSF), other non-respiratory sterile fluid or tissue	1.0 ml (if applicable)	Sterile container

Submission and Collection Notes

Complete all fields of the COVID-19 and Respiratory Virus Test Requisition, including:

Test(s) requested and indication for testing (e.g. immunocompromised)*
Clinical information including symptom onset date*
Medical condition that puts the patient at increased risk for severe illness *
Patient setting (e.g. ward, ICU, congregate living, Long-term care)*
Specimen type and collection date
Valid outbreak number (if applicable)
Travel history (if applicable)
COVID-19 vaccination status
* Mandatory information. If the patient is eligible for public funded testing as per the Ministry of Health testing guidance,this must be indicated on the requisition.
FAILURE TO PROVIDE THE ABOVE INFORMATION MAY RESULT IN REJECTION OR TESTING DELAY.

For clinical specimens, label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. For additional information see: Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay.

Number of specimen submissions
PHO will accept multiple swabs (e.g NP swab, throat swab) placed into one transport medium for hospitalized patients. This combined submission will be run and reported as a single test. Specimens can be from different collection sites as listed in the Specimen Collection Options table above.

Testing for other respiratory viruses
If patients meet testing criteria, Multiplex Respiratory Virus Panel (MRVP) or FLUVID testing can be ordered on the same specimen submitted for COVID-19 testing, except for saliva, which is not validated for seasonal respiratory virus testing. SARS-CoV-2 virus does not cross-react with other respiratory viruses in the MRVP assay. Refer to Respiratory Viruses (including influenza) Test Information for more details and acceptance criteria.

“Eligible healthcare providers can also order Covid-19 supplies through the Supply Ontario portal.”

Limitations

COVID-19 PCR testing is validated at PHO for testing of the respiratory specimen types described in the Specimen Collection Options table above. Non-respiratory specimens will be tested and reported with a disclaimer.

Storage and Transport

Place specimen in biohazard bag and seal. Specimens should be stored at 2-8°C following collection and shipped to PHO 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 a PHO location conducting COVID-19 PCR testing in accordance with the Transportation of Dangerous Goods Regulations. PHO testing locations include Toronto, London, and Thunder Bay.

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

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

COVID-19 testing is performed at PHO:

Toronto: Monday to Sunday
London and Thunder Bay: Monday to Saturday

Please continue to submit specimens for testing to your local public health laboratory, samples will be re-routed accordingly to meet the expected turn-around time.

Most results are expected to be completed within 24 hours, however turnaround time may vary according to geographical location and proximity to PHO testing locations.

Test Methods

Testing for COVID-19 is done by real-time reverse transcription PCR using the following validated assay:

Assay	Gene Targets
PHO laboratory developed test (LDT)	E gene*

E – Envelope

*Based on Corman et. al (Euro Surveill 2020).

Interpretation

The following table provides possible test results with associated interpretations:

Result	Interpretation	Comments
Not Detected	Specimens with no gene target(s) detected will be reported as COVID-19 virus not detected. The absence of detected gene target(s) is sufficient to exclude a COVID-19 diagnosis in many situations.
Detected	Specimens with a single gene target detected will be reported as COVID-19 virus detected.
Detected (Low Level)	Specimens with E gene target detected at a late amplification signal within a predetermined high cycle threshold (Ct) value range (Ct ≥ 35 and ≤ 38 for PHO’s LDT) will be reported as COVID-19 virus (low level) detected. The low level detection of any gene target is sufficient to confirm a COVID-19 diagnosis in most situations.	The low level positivity may further correlate with either a very early or very late stage of the COVID-19 infection, and this additional information may assist public health units in defining transmission risk.
Indeterminate	Specimens with a gene target detected at a very late amplification signal within a predetermined high cycle threshold (Ct) value range (Ct > 38 and <40 for PHO’s LDT) will be reported as COVID-19 virus indeterminate. An indeterminate result may be due to very low viral target quantity in the clinical specimen approaching the limit of detection of the assay, or may represent nonspecific reactivity (false signal) in the specimen.
Invalid	Tests may be reported as invalid if gene target results are uninterpretable. This could be for various reasons, most commonly due to failed detection of the assay’s internal 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.

Reporting

Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

Specimens that are positive for COVID-19 are reported to the medical officer of health as per Health Protection and Promotion Act

References

Tsang NNY, So HC, Ng KY, Cowling BJ, Leung GM, Ip DKM. Diagnostic performance of different sampling approaches for SARS-CoV-2 RT-PCR testing: a systematic review and meta-analysis. The Lancet Infectious Diseases [Internet]. 2021 Apr 12 [cited 2021 Apr 24];0(0). Available from: https://www.thelancet.com/journals/laninf/article/PIIS1473-3099(21)00146-8/fulltext
Kandel CE, Young M, Serbanescu MA, Powis JE, Bulir D, Callahan J, et al. Detection of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) in outpatients: A multicenter comparison of self-collected saline gargle, oral swab, and combined oral–anterior nasal swab to a provider collected nasopharyngeal swab. Infection Control & Hospital Epidemiology. 2021 Jan 13;1–5.
Jamal AJ, Mozafarihashjin M, Coomes E, Anceva-Sami S, Barati S, Crowl G, et al. Sensitivity of midturbinate versus nasopharyngeal swabs for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection Control & Hospital Epidemiology. 2020 Nov 18;42(8):1001–3.
Butler-Laporte G, Lawandi A, Schiller I, Yao M, Dendukuri N, McDonald EG, et al. Comparison of Saliva and Nasopharyngeal Swab Nucleic Acid Amplification Testing for Detection of SARS-CoV-2. JAMA Internal Medicine [Internet]. 2021 Mar 1 [cited 2021 Mar 16];181(3):353. Available from: https://jamanetwork.com/journals/jamainternalmedicine/fullarticle/2775397
Kandel C, Zheng J, McCready J, Serbanescu M, Racher H, Desaulnier M, et al. Detection of SARS-CoV-2 from Saliva as Compared to Nasopharyngeal Swabs in Outpatients. Viruses. 2020 Nov 17;12(11):1314
Goldfarb DM, Tilley P, Al-Rawahi GN, Srigley JA, Ford G, Pedersen H, et al. Self-Collected Saline Gargle Samples as an Alternative to Health Care Worker-Collected Nasopharyngeal Swabs for COVID-19 Diagnosis in Outpatients. Tang Y-W, editor. Journal of Clinical Microbiology. 2021 Mar 19;59(4).
Kojima N, Turner F, Slepnev V, Bacelar A, Deming L, Kodeboyina S, et al. Self-Collected Oral Fluid and Nasal Swab Specimens Demonstrate Comparable Sensitivity to Clinician-Collected Nasopharyngeal Swab Specimens for the Detection of SARS-CoV-2. Clinical Infectious Diseases. 2020 Oct 19;
Teo AKJ, Choudhury Y, Tan IB, Cher CY, Chew SH, Wan ZY, et al. Saliva is more sensitive than nasopharyngeal or nasal swabs for diagnosis of asymptomatic and mild COVID-19 infection. Scientific Reports. 2021 Feb 4;11(1).
Lee RA, Herigon JC, Benedetti A, Pollock NR, Denkinger CM. Performance of Saliva, Oropharyngeal Swabs, and Nasal Swabs for SARS-CoV-2 Molecular Detection: A Systematic Review and Meta-analysis. Journal of Clinical Microbiology. 2021 Jan 27;
Gertler M, Krause E, van Loon W, Krug N, Kausch F, Rohardt C, et al. Self-collected oral, nasal and saliva samples yield sensitivity comparable to professional-collected oro-nasopharyngeal swabs in SARS-CoV-2 diagnosis among symptomatic outpatients. International Journal of Infectious Diseases. 2021 Jul;
Grijalva CG, Rolfes M, Zhu Y, Chappell J, Halasa N, Kim A, et al. Performance of Self-Collected Anterior Nasal Swabs and Saliva Specimens for Detection of SARS-CoV-2 During Symptomatic and Asymptomatic Periods. Open Forum Infectious Diseases. 2021 Sep 25;8(11).
Marais G, Hsiao N, Iranzadeh A, Doolabh D, Enoch A, Chu C, et al. Saliva swabs are the preferred sample for Omicron detection. 2021 Dec 24;
Schrom J, Marquez C, Pilarowski G, Wang C-Y, Mitchell A, Puccinelli R, et al. Comparison of SARS-CoV-2 Reverse Transcriptase Polymerase Chain Reaction and BinaxNOW Rapid Antigen Tests at a Community Site During an Omicron Surge. Annals of Internal Medicine. 2022 Mar 15;
Goodall BL, LeBlanc JJ, Hatchette TF, Barrett L, Patriquin G. Investigating the Sensitivity of Nasal or Throat Swabs: Combination of Both Swabs Increases the Sensitivity of SARS-CoV-2 Rapid Antigen Tests. Martin RM, editor. Microbiology Spectrum. 2022 Aug 31;10(4).

Data and Analysis

Related Testing Resources