Cryptosporidium or Cyclospora — Typing

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Background
This document provides testing information for Cryptosporidium and Cyclosopra typing (also known as genotyping or subtyping) at Public Health Ontario.

This page is specific to subtyping of known positive specimens. For primary testing of patients suspected of cryptosporidiosis or cyclosporiasis, please refer to the following test page: Enteric Protozoa (Cryptosporidium, Cyclospora, Dientamoeba, Entamoeba, and Giardia) – PCR.

Updates

Cryptosporidium typing is now included in this test information page.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 19 oct. 2026.

Testing Indications

All positive Cyclospora specimens should be sent to PHO for typing surveillance and to identify potential sources of outbreaks. Cyclospora typing is routinely performed on all unpreserved specimens positive for Cyclospora in Ontario. All cases identified on preserved specimens (e.g. SAF) should undergo repeat collection using unpreserved specimens as soon as possible to assist in public health investigations.

If requested by a local public health unit, Cryptosporidium specimens may be sent to PHO for typing to identify potential sources of outbreaks. Testing is only performed in the context of an established outbreak investigation.

Acceptance/Rejection Criteria

The following two criteria are required for typing to be performed:

Specimen is already known to be positive for Cryptosporidium or Cyclospora (e.g. by microscopy or molecular methods).
Specimen is unpreserved or in Cary-Blair transport medium.
For Cryptosporidium specifically: the specimen is accompanied by an investigation or outbreak number.

NOTE: specimens preserved in formalin or SAF are NOT acceptable for typing.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Cyclospora – Typing	General Test Requisition	Enteric specimen (e.g. stool, duodenal contents, tissue biopsy) positive for Cyclospora	0.5 g or 1ml	Cary-Blair vial or Sterile empty vial
Cryptosporidium – Typing	General Test Requisition	Enteric specimen (e.g. stool, duodenal contents, tissue biopsy) positive for Cryptosporidium	0.5 g or 1ml	Cary-Blair vial or Sterile empty vial

Submission and Collection Notes

Complete all fields of the requisition form.

Important: Specify which organism is already known to be positive (Cryptosporidium or Cyclospora) on the requisition.

Indicate if the patient has known travel history under ‘Recent Travel (Specify Location)’ field.

If the patient is part of a cluster/outbreak investigation, indicate the investigation or outbreak number in the “Public Health Outbreak No.” field or otherwise contact PHO’s Laboratory Customer Service at 416-235-6556/1-877-604-4567 prior to sample submission.

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. For additional information see: Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay.

Storage and Transport

Place specimen container in a biohazard bag and properly seal the bag. Specimens should be stored at 2-8°C and shipped to PHO on ice packs within 48 hours of collection. If specimens might not be able to arrive at PHO within 48 hours of collection, they should be stored frozen (- 20°C or lower) and shipped on ice pack as soon as possible. All specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Cryptosporidium and Cyclospora typing are forwarded to the National Microbiology Laboratory (NML) in Guelph. Results are made available to national and provincial epidemiologists for cluster investigations and outbreak response.

Test Methods

Method: Cyclospora typing is performed at NML using targeted amplicon deep sequencing (TADS) as per Yanta CA et al. (2022)¹ and based on a similar US Centers for Disease Control and Prevention (US CDC) protocol. Temporal-genetic cluster codes are assigned based on genotypic relatedness and date of collection for national response. Cryptosporidium typing is performed at NML using gp60 gene Sanger sequencing per Guy RA et al (2021).^2,3

Performance and Limitations: Sequencing methods are currently undergoing validation and used for epidemiological surveillance and outbreak investigations only. Results are not actionable for clinical management.

Interpretation

Results will be finalized as “Sample received for surveillance purposes, no further result to report” with no further results provided directly to submitters.

Reporting

At this time, sequencing results will only be made available to PHO and Public Health Agency of Canada (PHAC) epidemiologists, such as through the Canadian Network for Public Health Intelligence (CNPHI) for cluster investigations. A generic confirmation of receipt will be provided to the specimen submitter as indicated on the requisition.

References

Yanta CA, Barta JR, Corbeil A, Menan H, Thivierge K, Needle R, Morshed M, Dixon BR, Wasmuth JD, Guy RA. Genotyping Canadian Cyclospora cayetanensis Isolates to Supplement Cyclosporiasis Outbreak Investigations. Microorganisms. 2022 Feb 15;10(2):447. doi: 10.3390/microorganisms10020447. PMID: 35208901; PMCID: PMC8879297.
Robinson G, Chalmers RM, Elwin K, Guy RA, Bessonov K, Troell K, Xiao L. Deciphering a cryptic minefield: A guide to Cryptosporidium gp60 subtyping. Curr Res Parasitol Vector Borne Dis. 2025 Mar 27;7:100257. doi: 10.1016/j.crpvbd.2025.100257. PMID: 40256454; PMCID: PMC12008548.
Guy RA, Yanta CA, Muchaal PK, Rankin MA, Thivierge K, Lau R, Boggild AK. Molecular characterization of Cryptosporidium isolates from humans in Ontario, Canada. Parasit Vectors. 2021 Jan 22;14(1):69. doi: 10.1186/s13071-020-04546-9. PMID: 33482898; PMCID: PMC7821412.