SARS-CoV-2 (COVID-19 Virus) Genomic Surveillance

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

As of November 12, 2021 PHO Laboratory is no longer testing SARS-CoV-2 PCR positive specimens for N501Y and E484K mutations by multiplex real time PCR. This is due the current dominance of the B.1.617.2 (Delta) lineage in Ontario that has reduced the utility of the N501Y and E484K targets for differentiating circulating VOC lineages. A shift to a representative surveillance strategy real-time PCR testing for specific mutations in SARS-CoV-2 positive specimens will be revisited as needed.

SARS-COV-2 WHOLE GENOME SEQUENCING FOR SURVEILLANCE

As of November 12, 2021 PHO Laboratory will be randomly selecting a proportion (e.g. 25%) of all SARS-CoV-2 positive specimens, with Ct≤ 30* from the SARS-CoV-2 PCR diagnostic test, for whole genome sequencing (WGS). This replaces the previous process implemented on May 26, 2021 for sequencing based on the VOC real-time PCR test.

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

This link provides the latest summary for SARS-CoV-2 Whole Genome Sequencing in Ontario.

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 from cases that are positive for SARS-CoV-2 ≥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.

For criteria 1 and 2, sequencing results will be sent as a lab information system standard report 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 (VOIs) will not be identified as such on the laboratory report, nor will the lineage be reported. Descendent lineages of VOCs will be reported as the parent lineage only (e.g. AY.4 Delta descendent lineage reported as B.1.617.2).

For criteria 3 and 4, outbreak investigations, the submitter may request a simple lineage-only report or in-depth genomic analysis. If WGS testing is agreed to by the Ontario COVID-19 Genomic Network, submitters should confirm the specimen’s eligibility and availability with the original testing lab and arrange for specimens to be sent to the WGS testing laboratory. Please refer to the SARS-CoV-2 Whole Genome Sequencing Request guidance document and contact IORT@oahpp.ca for support.

* The specimen’s SARS-CoV-2 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 Genomic Surveillance

SARS-CoV-2 PCR-positive specimen

1 ml preferred (min 500 µl)

Samples should be submitted in screw cap tube (Simport or O-ring, etc) only. Do NOT submit samples in snap cap

Submission and Collection Notes

1

For travel, vaccinated and super spreading event, if the specimen is at PHO Laboratory call Customer Service Centre (416-235-6556 or 1-877-604-4567) or fax the SARS–CoV-2 Whole Genome Sequencing Requisition 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.

2

For outbreak investigations email IORT@oahpp.ca to complete the request form and obtain approval/WGS-request ID before shipping any specimens. Submitters will receive information on signing up for a secure Incident and Outbreak Response Collaboration SharePoint Site account. This site has been authorized for the transfer of Personal Health Information and submitters will receive the in-depth genomic reports via this site.

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 whole genome sequencing, 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.

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

Reporting

As a disease of public health significance, all SARS-CoV-2 whole genome sequencing results will be reported to the local public health unit and the ordering health-care provider as indicated on the requisition.

Test Methods

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

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 bioinformatic tools and assigned a Pango lineage using the PANGOLINsoftware package, allowing for the identification of VOC and other lineages.

Algorithm

  1. A proportion of all SARS-CoV-2 positive specimens with Ct ≤ 30 will be selected for sequencing
  2. Specimens not already selected for sequencing that meet select conditions (e.g. post-vaccine, international travel, outbreak) can be requested to be sequenced
  3. As of November 12, 2021, specimens will no longer be selected for sequencing (WGS) based on results of the multiplex VOC 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

Updated 12 Nov 2021