SARS-CoV-2 (COVID-19 Virus) Variant of Concern (VoC) Surveillance

SARS-COV-2 MUTATION TESTING BY REAL-TIME PCR

As of March 22, 2021, all COVID-19 PCR-positive specimens with Ct value ≤35* in Ontario will be tested for the N501Y and E484K mutations using a multiplex real-time PCR assay. This test replaces the N501Y single target test that was implemented February 3, 2021. These two mutations are present in some described Variants of Concern (VOCs) as follows:  

Variant of Concern

Mutation(s)1

B.1.1.7 (Alpha) (202012/01) (originally identified in the UK)

N501Y

B.1.351 (Beta) (501Y.V2) (originally identified in South Africa)

N501Y, E484K

P.1 (Gamma) (originally identified in Brazil)

N501Y, E484K

B.1.617.2 (Delta) (originally identified in India)

No N501Y or E484K mutations

1Only mutations detected by the VOC multiplex assay are listed.

Specimens that are N501Y positive and E484K negative are presumed to be likely B.1.1.7 (Alpha) and specimens that are negative for both N501Y and E484K are presumed to be likely B.1.617.2 (Delta) based on current epidemiology.

As of June 7, 2021 PHO Laboratory implemented a K417N/T VOC mutation PCR assay to provide timely differentiation between presumed B.1.351 (Beta) and P.1 (Gamma) lineages. P.1 (Gamma) VOCs will be positive for the K417T target, and B.1.351 (Beta) VOCs will be positive for the K417N target.

This assay can be requested to support outbreak investigations – testing up to 2 specimens is recommended for the purpose of clarifying the lineage causing an outbreak when all specimens are E484K and N501Y positive, suggesting a single lineage as the cause of the outbreak.

The K417 N/T VOC PCR may also be requested by healthcare provider teams in hospitals (e.g. IPAC, Infectious Diseases) to support management of hospitalized patients, in particular to facilitate cohorting decisions.

Specimens must be positive for both N501Y AND E484K mutations to be eligible for K417N/T testing.

All VOC PCR results (and subsequent sequencing results where applicable, see below) will be reported to the ordering healthcare provider; all mutation positive results, as well as all K417N/T VOC PCR results will be reported to the public health unit.

Ongoing universal VOC N501Y/E484K mutation testing of all PCR-positive specimens will be revisited as needed.
 

* The specimen’s SARS-CoV-2 cycle threshold (Ct) value must be 35 to ensure successful N501Y/E484K mutation testing.  A subset of samples with a CT ≤ 35 may not be successfully tested for VOC mutations likely due to a low level of virus present or RNA degradation.


SARS-CoV-2 VOC Genome Sequencing for Surveillance

As of May 26, 2021 PHO Laboratory changed the specimen selection process for whole genome sequencing (WGS) and will be randomly selecting a proportion of all COVID positive specimens with Ct≤ 30* from the VOC-PCR test (e.g. 10% of all positive specimens) regardless of the VOC-PCR result. This replaces the process implemented on March 22, 2021 for sequencing all E484K positives and a 5% selection of E484K negatives.

The proportion of selected specimens will be determined according to current positivity rates, the number of positive specimens, and testing capacity. When capacity allows, up to 100% of eligible specimens will be tested by WGS and was first implemented as of June 14, 2021. This sampling method will remove selection bias, provide better representation of known variants for surveillance, and allow improved detection of new emerging variants. 

In addition to the selected proportion (if less than 100%), sequencing will be performed on specimens meeting the following criteria:

  1. International travel – all specimens from travelers and contacts will be sequenced if travel is indicated on the COVID-19 diagnostic test requisition. If travel was not indicated on the requisition testing can be added upon request.
  2. Vaccine breakthrough – all specimens of cases that are positive for COVID-19 ≥14 days after the completion of the vaccination series (e.g.  ≥14 days after the second dose of a 2-dose series) will be sequenced if vaccination status is indicated on the COVID-19 diagnostic test requisition. If vaccination status was not indicated on the requisition testing can be added upon request.
  3. Suspected reinfection – sequencing can be requested to support the classification of the case as re-infection.
  4. Certain outbreak investigations – sequencing can be requested to support outbreak investigations.

Sequencing results will be reported back to the ordering healthcare provider and to the public health unit for all specimens, however only VOC lineages will be named on the report. Lineages not currently designated as VOC will be reported back as “Variant of Concern not detected” due to the dynamic nature of non-VOC lineage naming. Similarly, Variants of Interest (VOI) will not be identified as such on the laboratory report, nor will the lineage be reported.

* The specimen’s VOC mutation testing PCR cycle threshold (Ct) value must be ≤30 for successful genome sequencing.  A subset of samples with a CT ≤ 30 may not be successfully sequenced likely due to a low level of virus present, RNA degradation, or sequencing technical issues.

Specimen Collection and Handling

Specimen Requirements

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

SARS-COV-2 VARIANT SCREENING

SARS-CoV-2 PCR-positive specimen

1 ml preferred (min 500 µl)

N/A

Submission and Collection Notes

1

SARS-CoV-2 VOC testing cannot be performed prior to laboratory detection of SARS-CoV-2. All positive SARS-CoV-2 specimens will be tested for N501Y and E484K mutations. Additional SARS-CoV-2 VOC testing may be requested on specimens meeting acceptance criteria (see above).

2

To request K417N/T testing –

  1. specimens should meet the following criteria:
    1. Specimen is positive for both N501Y AND E484K mutations
    2. Specimen is part of an outbreak investigation or from a hospitalized patient
    3. Differentiation of B.1.351 (Beta) from P.1 (Gamma) lineages is required for outbreak or patient management
  2. If the specimen is at PHO Laboratory call Customer Service Centre (416-235-6556 or 1-877-604-4567) or fax the VOC Information Form to (416-235-6552) to request to add the test; if the specimen is at another laboratory use the form above to send the specimen to PHO Laboratory. Check-mark the “Other (specify)” box in Section 1 – Indication for SARS-CoV-2 Supplemental testing. In the field below “Other (specify)” indicate “N501Y+/E484K+ for K417N/T PCR”.

Limitations

This laboratory-developed investigational assay is being used for surveillance purposes and public health management. It is not a clinical test.

Preparation Prior to Transport

For SARS-CoV-2-positive specimens being sent to PHO Laboratory for VOC mutation testing, 1 mL (minimum volume 500 µL) of specimen should be provided and maintained at refrigeration temperature during transportation (freeze if anticipated transport time to the laboratory is >72 hours).

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Surveillance testing will be performed at regular intervals.

SARS-CoV-2 VOC Multiplex real-time PCR results will be available within 3 days.

SARS-CoV-2 PCR assay to detect K417N/T mutation will be available upon request for specimens that meet acceptance criteria outlined above. Results will be available within 48 hours of request.

If performed, SARS-CoV-2 genome sequencing results will be available within 2-4 weeks.

Reporting

All SARS-CoV-2 VOC testing results are reported back to the ordering health-care provider as indicated on the requisition.

As a disease of public health significance, detection of SARS-CoV-2 VOC-related mutations and VOCs will be reported to the local public health unit.

Test Methods

This laboratory-developed investigational assay is being used for surveillance purposes and public health management.

Testing involves a multiplexed single nucleotide polymorphism (SNP) real-time PCR assay developed at PHO Laboratory. The assay screens for the N501Y and E484K mutations in the spike (S) gene, which are associated with VOCs (see table below). To further differentiate between B.1.351 (Beta) and P.1 (Gamma) lineages PHO Laboratory implemented a second PCR test that detects K417N/T. This will be an add-on test in cases where this differentiation can support public health action. Testing will be restricted to outbreak investigations and IPAC measures in hospitalized patients.

Test result interpretation for VOC-PCR testing: 

VOC multiplex result

K417N/T SNP PCR result

Presumed VOC

Comments

N501 wild type

Not Applicable

Not a VOC with mutations at amino acids 484 or 501; could be another VOC (e.g. B.1.617.2 [Delta])

 

N501Y positive; E484K negative

Not Applicable

B.1.1.7 (Alpha)

 

N501Y positive and E484K positive

K417N positive

B.1.351 (Beta)

 

N501Y positive and E484K positive

K417T positive

P.1 (Gamma)

 

N501Y positive and E484K positive

K417N/T negative

Could be B.1.1.7  (Alpha) with E484K, or not a VOC.
See comment

K417N/T mutations may be present but not detected in specimens with low viral load (e.g. Ct ≥30) due to limited assay sensitivity.

N501Y positive and E484K positive

Not done

B.1.351 (Beta) or P.1 (Gamma)

PCR test for K417N/T not routinely done. Test is only performed in specific cases to support patient management. See above for details.

N501Y positive and E484K positive

Invalid

B.1.351 (Beta) or P.1 (Gamma)

Results uninterpretable due to failed amplification of extraction control. Unable to report 417 N/T assay result. Amplification failure may be due to inadequate specimen content, extraction failure, or  PCR inhibition


Sequencing involves PCR amplification, followed by sequencing of the majority of the approximately 30,000 base pair SARS-CoV-2 genome. Generated sequences are processed using bioinformatics analysis and assigned a Pango lineage using the pangolin tool, allowing for the identification of VOC and other lineages.

Algorithm

  1. SARS-COV-2 positive specimen with Ct≤35 are tested by multiplex real-time PCR assay for N501Y and E484K mutations
  2. A proportion of all positive specimens with VOC PCR test with ct < 30 (regardless of VOC PCR result) will be selected for sequencing
  3. Specimens not already selected for sequencing with both N501Y AND E484K mutations DETECTED, can be requested to be tested by the K417N/T VOC PCR assay to differentiate between the B.1.351 and P.1 lineages (restrictions apply)
  4. Specimens not already selected for sequencing that meet select conditions (e.g. post-vaccine, international travel, outbreak) can be requested to be sequenced
  5. Specimens will no longer be selected for sequencing (WGS) based on results of multiplex real-time PCR assay

Data and Analysis

Related Testing Resources

PHO has developed the following testing resources:

Frequently Asked Questions: SARS-COV-2 (COVID-19 virus) Variant of Concern (VOC) Testing Update, March 26, 2021

Mis à jour le 10 juill. 2021