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:  

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.