Streptococcus pyogenes (Group A Streptococcal Disease or GAS) – Typing

Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.

Background
This page provides supplementary testing information on Group A streptococcal (GAS) disease typing at Public Health Ontario (PHO). The causative agent of GAS disease is Streptococcus pyogenes.

This page is limited to supplementary typing information of confirmed GAS cases to assist with surveillance and cluster investigations. For information regarding routine bacterial identification and/or confirmation of cultured Streptococcus pyogenes isolates, refer instead to Bacterial cultures- Aerobic- Reference ID/Confirmation.

Updates
As of November 13, 2023 this testing information replaces previously published testing information pages for:

GAS-Group A Streptococcus Outbreak
Group A Streptococcus Serotyping
GAS-Group A Streptococci -Pulse Field Gel Electrophoresis

Information from these previous testing pages are now consolidated onto this Streptococcus pyogenes (Group A Streptococcal Disease or GAS) testing page.

*Uncontrolled print copy. Valid only on day of print: 01 May 2024.

Testing Indications

Routine M protein gene (emm) typing is recommended for all cases of invasive GAS (iGAS) in Ontario in order to assist with disease surveillance. Cases of iGAS include:

S. pyogenes isolated from a sterile site (e.g. blood, cerebrospinal fluid, joint fluid, pleural fluid, pericardial fluid), or
S. pyogenes isolated from a non-sterile site (e.g. skin) with evidence of disease severity (e.g. necrotizing fasciitis, streptococcal toxic shock syndrome, meningitis).

Both emm typing and detailed cluster phylogenetic analysis is available upon request for cases of GAS from patients who are part of an outbreak investigation by a health unit if meeting the following criteria:
- Outbreak number assigned by the health unit, and
- Index case’s cultured isolate available, and
- Historical isolates that are part of the outbreak also available. Note: if these were not previously submitted to PHO’s laboratory, the health unit should attempt to locate them at the original testing laboratory and arrange for the holding laboratory to transfer the cultured isolates to PHO’s laboratory with the outbreak investigation number included.
Both emm typing and detailed cluster phylogenetic analysis is available upon request for cases of GAS from patients who are part of a cluster (non-outbreak) investigation if meeting the following criteria:
- Temporal increase in iGAS in a geographic area or suspicion for a GAS cluster in an institution that does not meet the provincial outbreak case definitions, and
- Analysis pre-approved by the PHO microbiologist, and
- Investigation number assigned by PHO.

As a Disease of Public Health Significance (DOPHS), additional recommendations are available in the Ontario Public Health Standards Appendix on iGAS.

Acceptance/Rejection Criteria

Any of the following conditions will result in test rejection:

Primary specimen* (e.g. skin swab, blood, CSF), or
Mixed or non-viable cultured isolate, or
Mislabelled or un-labelled cultured isolate, or
Cultured isolates from a non-sterile site for which the disease severity is not clearly stated on the requisition, or
Cultured isolates for cluster/outbreak investigation analysis without an associated number on the requisition.

*Note: Only pure and viable cultured isolates are accepted for testing. Primary specimens are not processed at PHO’s laboratory and will be rejected. Primary specimens should be initially processed at a community laboratory or local hospital laboratory.

Specimen Collection and Handling

Specimen Requirements

Test Requested

Required Requisition(s)

Specimen Type

Minimum Volume

Collection Kit

Streptococcus pyogenes, group A strep, or GAS typing or cluster analysis

Reference Bacteriology Requisition

Pure viable cultured isolate of Streptococcus pyogenes

N/A

Solid blood agar medium (or any non-selective solid agar that supports organism growth)

Semi-solid Amies charcoal transport medium swab

Submission and Collection Notes

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. Failure to provide this information may result in rejection or testing delay.

Please be sure to complete all fields on the Reference Bacteriology Requisition including:

Test requested
Specimen source
Isolate information (including gram stain, catalase, oxidase)
Isolate identification
Clinical/epidemiology information
Date of primary specimen collection
Primary specimen source (mandatory)
If the primary specimen source is blood: number of consecutive blood cultures positive for the submitted isolate
If applicable: outbreak or investigation number

Only submit one isolate per patient (NOT per specimen source). If the organism was isolated from multiple specimen sources, the order of preference of specimen submission is CSF first, otherwise blood, otherwise any other sterile site.

PHO’s laboratory does not provide swabs for primary GAS outbreak screening nor tests for GAS from primary specimens.

If the patient is part of a cluster/outbreak investigation, contact PHO’s laboratory Customer Service at 416-235-6556/1-877-604-4567 prior to sample submission.

Limitations

If a Streptococcus pyogenes cultured isolate is submitted in a semi-solid Amies charcoal transport medium swab as opposed to a solid agar plate, the turnaround time (TAT) may be increased by at least 24 hours to accommodate sub-culturing.

Storage and Transport

Cultured isolates must be stored at 2-8°C if they cannot be shipped to PHO’s laboratory on the same day that the incubation is completed. Refrigerated (2-8°C) cultured isolates must be shipped to PHO’s laboratory on ice packs within 3 days. All cultured isolates must be shipped in accordance to the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Streptococcus pyogenes typing is forwarded to the National Microbiology Laboratory (NML) once per week for emm genetyping surveillance and/or cluster phylogenetic analysis. More frequent submissions are available from Monday to Thursday upon request for cluster investigation samples.

Turnaround time for emm gene typing is up to 30 days from receipt at the NML. Priority testing for detailed cluster phylogenetic analysis may be expedited within two weeks from receipt at PHO’s laboratory upon request.

STAT and Critical Samples Testing

Priority testing for cluster/outbreak investigations is available upon request. If needed, contact PHO’s laboratory Customer Service at 416-235-6556/1-877-604-4567 prior to sample submission.

Test Methods

Cultured isolates received at PHO’s laboratory will be confirmed as Streptococcus pyogenes by biochemical testing and/or mass spectrometry prior to typing.

emm gene typing: Typing is performed by the NML using a whole genome sequencing (WGS) method.¹ In addition to WGS, targeted emm gene Sanger sequencing may be performed first by NML upon request to expedite results for priority testing.²

Cluster phylogenetic analysis: Cluster analysis is performed by the NML using a WGS method combining emm typing, multilocus sequence typing (MLST), and single nucleotide polymorphism (SNP) analyses of the sequences obtained.³ In addition to WGS at NML, a traditional pulse-field gel electrophoresis (PFGE) method with SmaI macrorestriction can also be performed in-house by PHO upon request for priority testing.⁴

Performance and Limitations
emm gene typing:WGS provides the highest resolution and predictive value to assign emm types compared to other emm typing methods such as Sanger sequencing. De novo WGS also allows for the detection of non-validated emm types and dual emm/emm-like pairs. However, Sanger sequencing may provide faster preliminary results for timely cluster investigations.

Cluster phylogenetic analysis: WGS provides the highest resolution and predictive value to assign genetic relatedness compared to other phylogenetic analysis methods such as PFGE. PFGE may infer high similarity between isolates but may overcall relatedness due to the limited number of sequence types evaluated.

Algorithm

All iGAS isolates: routine emm typing performed on submitted isolates (one isolate per patient).
Outbreak/cluster investigation isolates: emm typing performed on submitted isolates meeting the testing indication above. Additional cluster phylogenetic analysis only available upon request.

Reporting

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

Outbreak testing results and significant results from investigation are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

References

Kapatai G, Coelho J, Platt S, Chalker VJ. Whole genome sequencing of group A Streptococcus: development and evaluation of an automated pipeline for emmgene typing. PeerJ. 2017 Apr 27;5:e3226. doi: 10.7717/peerj.3226.
Beall B, Facklam R, Thompson T. Sequencing emm-specific PCR products for routine and accurate typing of group A streptococci. J Clin Microbiol. 1996 Apr;34(4):953-8. doi: 10.1128/jcm.34.4.953-958.1996. PMID: 8815115; PMCID: PMC228924.
Tagini F, Aubert B, Troillet N, Pillonel T, Praz G, Crisinel PA, Prod'hom G, Asner S, Greub G. Importance of whole genome sequencing for the assessment of outbreaks in diagnostic laboratories: analysis of a case series of invasive Streptococcus pyogenes infections. Eur J Clin Microbiol Infect Dis. 2017 Jul;36(7):1173-1180. doi: 10.1007/s10096-017-2905-z.
AStanley J, Linton D, Desai M, Efstratiou A, George R. Molecular subtyping of prevalent M serotypes of Streptococcus pyogenes causing invasive disease. J Clin Microbiol. 1995 Nov;33(11):2850-5. doi: 10.1128/jcm.33.11.2850-2855.1995.