Enteric Protozoa (Cryptosporidium, Cyclospora, Dientamoeba, Entamoeba, and Giardia) – PCR

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Background
This page provides routine PCR testing information for the main enteric parasitic protozoa at Public Health Ontario (PHO). These parasitic protozoa include Cryptosporidium, Cyclospora, Dientamoeba, Entamoeba, and Giardia.

For testing of other enteric parasites, refer to the following webpages:

Updates
This new webpage reflects the adoption of PCR as the primary methodology for enteric protozoa testing, replacing microscopy at Public Health Ontario.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 17 déc. 2025.

Testing Indications

Enteric protozoa PCR is indicated for the diagnosis of patients with unexplained diarrhea lasting for 7 days or longer associated with recent travel, untreated water exposure (e.g. well water or streams), impaired immune system, or a suspected outbreak cluster.¹

Note: For cases of extraintestinal amebiasis caused by Entamoeba histolytica, microscopy and PCR may be performed on non-fecal specimens (e.g. liver, brain, or lung abscess aspirates), in conjunction with serological testing. For detailed instructions, refer to the following PHO webpage: Extraintestinal Entamoeba histolytica (Amebic Abscess) – Microscopy, PCR, and Antibody.

Acceptance/Rejection Criteria

To ensure appropriate testing and accurate results, the following criteria must be met. Specimens that do not meet these requirements will be rejected.

Accepted if the requisition includes one of the following:

Signs or symptoms consistent with enteric protozoal infection (e.g. diarrhea)
Close contact of an infected person (e.g., household member or outbreak cluster)

Rejected if:

Specimens are received in sodium acetate, acetic acid, and formalin (SAF) vials. These are not compatible with PCR testing at PHO and will be cancelled.
Specimens are from non-human sources such as animals (e.g. pets) or environmental (e.g. food or water)

Note: Submit only one specimen per patient per clinical episode. Additional specimens will not be routinely tested, as a single specimen is typically sufficient for protozoa PCR testing.

Specimen Collection and Handling

Specimen Requirements

Test Requested

Required Requisition(s)

Specimen Type

Minimum Volume

Collection Kit

Enteric protozoa- PCR

Cryptosporidium-PCR

Cyclospora-PCR

Dientamoeba -PCR

Entamoeba- PCR

or Giardia - PCR

General Test Requisition

Stool

1.0 ml

Sterile empty vial

Cary-Blair vial: Faeces Enteric Bacteriology Kit Order# 390049

Submission and Collection Notes

Complete all fields of the requisition form.

Important: Specify which organism(s) are suspected on the requisition, if known.

Important: Specify any of the following testing indications on the requisition. Failure to provide this information will result in rejection:

Signs or symptoms compatible with enteric protozoal infection (e.g. diarrhea)
Close contact of an infected person (e.g. household member or outbreak cluster)

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.

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.

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)

PCR is performed daily from Monday to Friday at PHO’s Toronto and Ottawa laboratory sites. Turnaround time is up to 5 calendar days from receipt at PHO.

Test Methods

PCR is performed at PHO using the commercial Seegene Allplex GI-Parasite PCR multiplex assay. The assay detects Cryptosporidium, Cyclospora, Dientamoeba fragilis, Entamoeba histolytica, and Giardia duodenalis.

Performance and Limitations:
When compared retrospectively on known positive enteric specimens, the Seegene Allplex GI-Parasite sensitivity has been reported to range between 59-90% (Cyclospora), 87-95% (E. histolytica), 95-99% (Cryptosporidium), 96-99% (D. fragilis), and 97-99% (Giardia). When compared prospectively, the assay has equivalent or superior performance to two or more stool microscopic examinations, therefore one stool specimen is sufficient for protozoa PCR. Specificity is usually reported above 98% overall. The current protozoa PCR assay does not rule out infection or co-infection with other parasitic organisms (e.g. Cystoisospora, Sarcocystis, Balantioides, helminths, microsporidia). Residual DNA may remain detectable for weeks after infection and does not distinguish active from resolved infection. ^1-13

Algorithm

Cryptosporidium, Cyclospora, Dientamoeba fragilis, Entamoeba histolytica, and Giardia duodenalis are routinely tested. Blastocystis is not routinely tested or reported due to its lack of documented pathogenicity and increasing recognition of its role as a positive biomarker of healthy outcomes.14

Specimens positive for Cyclospora will be automatically forwarded to NML for genotyping following this PHO webpage: Cyclospora – Subtyping.

Interpretation

Result Per PCR Target	Interpretation
Detected	Detectable DNA for the specified organism. If multiple organisms are detected, clinical correlation is required to distinguish relevance of each organism in the patient’s clinical presentation.
Not Detected	No detectable DNA for the specified organism.
Invalid	Test results are invalid due to the failed amplification of the extraction control. Amplification failure may be due to inadequate specimen content, extraction failure, or PCR inhibition. Please resubmit another specimen for testing if clinically indicated.

Notes:

The role of Dientamoeba fragilis in illness is not fully defined. Evidence-based clinical management and exclusion of other aetiologies is strongly recommended if D. fragilis is detected by PCR.
This PCR assay does not test for Cystoisospora, Balantioides, helminths, or microsporidia. If those organisms are suspected, refer to our test information index for instructions.

Reporting

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

In addition, Cryptosporidium, Cyclospora, Entamoeba histolytica, and Giardia positive results are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

References

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