Coronavirus Disease 2019 (COVID-19) - Variant of Concern Screening and Whole Genome Sequencing Surveillance

This document provides testing information for COVID-19 variant of concern (VOC) screening by PCR as well as COVID-19 lineage surveillance by whole genome sequencing (WGS) at Public Health Ontario (PHO). The causative agent for COVID-19 disease is the SARS-CoV-2 virus.

The test information described below is limited to COVID-19 variant screen and surveillance testing. For other testing modalities, please refer to the following links:

Routine COVID-19 PCR testing: Public Health Ontario Coronavirus Disease 2019 (COVID-19) – PCR Test Information
COVID-19 serology testing: Public Health Ontario Coronavirus Disease 2019 (COVID-19) – Serology Test Information
COVID-19 antigen testing: Ontario Ministry of Health COVID-19 Clinical Assessments and Testing – Rapid Antigen Tests Information

Who to test:
COVID-19 VOC screening by PCR is only available by request when required for clinical or public health management. Testing is not routinely recommended and variant screening is not recommended for treatment decision making.
WGS may be performed on targeted specimens upon request (see details below). Ongoing COVID-19 lineage surveillance by WGS is performed on a subset of all specimens positive for COVID-19 by routine PCR across Ontario. The proportion of positive specimens selected for WGS may vary depending on operational capacity.

Updates (May 2022):
VOC screen testing indications have been clarified to reflect that it is only performed upon request.

SARS-CoV-2 VOC S Gene Target Failure Screening By Real-Time PCR

PHO has used the spike (S) gene target failure (SGTF) pattern on the Thermo Fisher TaqPath™ PCR assay from December 6, 2021 onward to screen for sublineages of the B.1.1.529 (Omicron) VOC. The SGTF pattern predicts a mutation called V69del/H70del.

SGTF is a feature of the BA.1 and BA.3 sublineages of the B.1.1.529 (Omicron) VOC, and several other Omicron sublineages, as well as the earlier B.1.1.7 (Alpha) variant that is no longer routinely circulating in Ontario. Other non-VOC lineages, including variants of interest (VOIs), may also possess this pattern.

Omicron sublineage BA.2 does not possess the V69del/H70del mutation and therefore does not display the SGTF pattern. Most other lineages currently circulating at very low levels in Ontario, including B.1.617.2 (Delta) and several VOIs also do not exhibit the SGTF pattern.

SARS-CoV-2 Whole Genome Sequencing for Surveillance

All clinical COVID-19 testing laboratories in the province participate in the WGS surveillance process. A proportion of positive COVID-19 PCR samples across the province are sequenced for population-wide surveillance of SARS-CoV-2 lineages circulating in Ontario. PHO provides weekly epidemiological summaries of surveillance data from SARS-CoV-2 Whole Genome Sequencing in Ontario.

The proportion of selected positive specimens is adjusted periodically and determined according to provincial positivity rates, the number of positive specimens over time, and lab network testing capacity. When capacity allows, up to 100% of eligible positive specimens in Ontario 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.

In addition to the selected proportion of positive specimens for WGS (if less than 100%), WGS will be performed on all positive specimens from international travelers and contacts if travel is indicated on the COVID-19 and Respiratory Virus Test Requisition.

There are also specific scenarios where WGS can be conducted by request:

International travel – WGS can be requested if not originally indicated on the test requisition.
Suspected reinfection – sequencing can be requested to support the classification of a case as reinfection if strongly suspected and if the case does not meet the time-based reinfection criteria as outlined in the Ontario COVID-19 Case Definition.
Certain outbreak investigations – WGS can be requested to support outbreak investigations where the local public health unit is trying to understand the dynamics of the outbreak in conjunction with the epidemiology.

For sequencing by request, 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.

As of December 23, 2021, all positive specimens indicating suspected vaccine breakthrough are no longer routinely sequenced for targeted WGS surveillance. Vaccine breakthrough will continue to be monitored by sequencing through specimens identified as such in the proportional sampling described above.

*Uncontrolled print copy. Valid only on day of print: 22 May 2022.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
SARS-COV-2 Variant of Concern PCR Screening	SARS–CoV-2 Variant of Concern Testing / Whole Genome Sequencing Requisition	SARS-CoV-2 PCR positive specimen with Ct value ≤ 35	1.5 ml preferred (min 1.0 ml)	NA
SARS-COV-2 Whole Genome Sequencing	SARS–CoV-2 Variant of Concern Testing / Whole Genome Sequencing Requisition	SARS-CoV-2 PCR positive specimen with Ct value ≤ 30	1.5 ml preferred (min 0.5 ml)	NA

Submission and Collection Notes

For VOC PCR screening requests, accompanying requisitions should indicate “VOC mutation PCR testing requested” and justification for such request in “Other (specify)” or “Outbreak / suspected super spreading event” for outbreak specimens to document the reason for requesting VOC PCR testing. In addition, enter the outbreak number under “Investigation or Outbreak No.”

For travel or suspected reinfection WGS requests, if the specimen is at PHO’s laboratory, call Customer Service Centre (416-235-6556 or 1-877-604-4567) or fax the SARS–CoV-2 Variant of Concern Testing/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.

For outbreak investigation WGS requests email IORT@oahpp.ca to complete the request form and obtain approval/request ID before shipping any specimens. Submitters will receive instructions on how to sign up an account on the secure Incident and Outbreak Response Collaboration SharePoint Site. 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

VOC PCR screening and WGS are laboratory-developed investigational assays being used for surveillance purposes and public health management only. These assays are not clinical tests.

For VOC screening PCR, only positive samples with cycle threshold (Ct) value ≤ 35 on the routine COVID-19 PCR test result are eligible.

For WGS surveillance, only positive samples with Ct values ≤ 30 on the routine COVID-19 PCR test result are eligible. A subset of samples with Ct values ≤ 30 may still not be successfully sequenced due to variability in determination of Ct values, RNA degradation, or sequencing technical issues.

Preparation Prior to Transport

For SGTF screening, 1.5 mL (minimum volume 1.0 mL) of specimen should be provided and maintained at 2-8°C during transportation (freeze if anticipated transport time to the laboratory is >72 hours).

For WGS testing, 1.5 mL (minimum volume 0.5 mL) of specimen should be provided and maintained at 2-8°C during transportation (freeze if anticipated transport time to the laboratory is >72 hours).

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

VOC PCR screening: will only be tested upon request on specimens that meet acceptance criteria outlined above. Results will be available within 48 hours of receiving the sample at PHO’s laboratory.

WGS surveillance: results will be available within 1-2 weeks of receiving the sample at PHO’s laboratory.

Reporting

All VOC PCR and WGS results will be reported to the ordering healthcare provider and the local Public Health Unit.

Sequencing results will be sent as a standard report back to the ordering healthcare provider and to the public health unit; however, only VOC lineages will be named on the report. Due to the dynamic nature of lineage naming, lineages not currently designated as VOC will be reported back as “Variant of concern not detected”. Similarly, variants of interest (VOIs) will not be identified as such on the laboratory report, nor will the lineage be reported. Descendent sublineages of VOCs will be reported as the parent lineage only (e.g., Omicron sublineage BA.1 reported as “B1.1.529 [Omicron]”, Delta sublineage AY.4 reported as “B.1.617.2 [Delta]”, etc.).

For sequencing by request, the submitter may request a simple lineage-only report or in-depth genomic analysis. Please refer to the SARS-CoV-2 Whole Genome Sequencing Request guidance document and contact IORT@oahpp.ca for support.

Test Methods

VOC screening by PCR is done using the Thermo Fisher TaqPathTM assay. The mechanism behind the SGTF pattern is based on some sublineages having a deletion at amino acid positions H69 and V70 within the spike protein (annotated as H69del/V70del). The TaqPathTM PCR assay has significantly reduced S gene target detection when the H69del/V70del mutation is present.

When using the TaqPath™ assay, a negative S gene target in the presence of other positive gene targets is considered a SGTF pattern suggestive of a SARS-CoV-2 variant with the H69del/V70del mutation. This pattern is assay-dependent, meaning that other assays having a negative S gene target in the presence of other positive gene targets does not necessarily mean the presence of the mutation. For a list of assays other than TaqPath™ that can use the SGTF pattern to predict the H69del/V70del mutation, you may refer to the FDA section on Tests with Detection Patterns That May be Associated with the SARS-COV-2 Omicron Variant.

Combined VOC PCR and WGS data from Ontario during the Dec 2021 – Jan 2022 winter holiday season has confirmed that a SGTF result is a reliable proxy for BA.1 sublineage screening (98.9% sensitivity, 99.9% specificity, 99.5% positive predictive value, 99.7% negative predictive value). Since December 20, 2021 , WGS is no longer being performed on all SGTF positive specimens to confirm the sublineage. A subset of all eligible SARS-CoV-2 positive specimens continue to be sequenced in line with the provincial representative sampling proportion.

WGS 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 PANGOLIN software package, allowing for the identification of VOCs and other lineages.

Algorithm

SGTF screening: Due to changes in the provincial testing strategy and to optimize the capacity of our laboratory and turnaround time, PHO discontinued routine VOC PCR screening of all positive samples in the province on December 30, 2021 but continued routine targeted VOC PCR screening of all specimens submitted from hospitalized patients as well as at least two specimens from each COVID-19 outbreak.

On January 26, 2022, PHO discontinued routine VOC PCR screening of outbreak specimens considering the predominance of the SGTF pattern in outbreak specimens (present in 98% of specimens at the time). On March 2, 2022, routine VOC PCR screening was also discontinued for hospitalized patients for similar reasons. VOC PCR screening is now only available upon request (see Who to test above ).

WGS surveillance: As of May 26, 2021, PHO performed WGS surveillance on a subset of samples from the VOC PCR screening test. On November 12, 2021, the WGS selection process was expanded to randomly select a proportion of all specimens positive on the routine COVID-19 PCR (as opposed to the VOC screening PCR test).

Interpretation

Table 1. SGTF VOC result interpretation at PHO’s laboratory.

VOC Real-Time PCR Result			S Gene Target Failure	Interpretation	Additional Notes
Orf1a/b gene	N gene	S gene
Detected with Ct value ≤ 30	Detected with Ct value ≤ 30	Not Detected	Present	Result consistent with SARS-CoV-2 S gene target failure (SGTF) due to a deletion at amino acid positions 69-70.
Detected with Ct value ≤ 30	Detected with Ct value ≤ 30	Detected	Absent	No evidence of SARS-CoV-2 S gene target failure (SGTF).	At this time, the absence of SGTF may be consistent with either BA.2 sublineage of B.1.1.529 (Omicron), B.1.617.2 (Delta), or other non-VOC lineages.
Detected with Ct value > 30	Detected with Ct value > 30	Not Detected	Inconclusive	Inconclusive for SARS-CoV-2 S gene target failure (SGTF) due to low viral load.	The lack of S gene detection may be due to low viral load in the specimen and is not necessarily due to a deletion at amino acid positions 69-70.
Not Detected	Detected	Not Detected	Unable to complete	Unable to complete	Unable to complete SARS-CoV-2 S gene target failure (SGTF) screening due to failure to detect one or both of the two additional gene targets in the assay (N or Orf1ab). Detection of both N and Orf1ab gene targets is required for documentation of SGTF
Detected	Not Detected	Not Detected	Unable to complete	Unable to complete	See above
Failed to detect internal control	Invalid	Invalid	Test results are uninterpretable due to failed detection of internal control(s). Please resubmit another specimen for testing if indicated.

* Based on current epidemiology, SARS-CoV-2 S gene target failure (SGTF) is consistent with the BA.1 and BA.3 sublineages of B.1.1.529 (Omicron) and rarely with B.1.1.7 (Alpha) or other lineages (including variants of interest [VOIs]). The lack of the SGTF pattern is currently consistent with either the BA.2 sublineage of B.1.1.529 (Omicron), B.1.617.2 (Delta), and rarely with other lineages.

Data and Analysis

*Uncontrolled print copy. Valid only on day of print: 22 May 2022.

Updated 18 May 2022