Entamoeba histolytica (Amebiasis) – Microscopy, Antigen, and PCR

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Background
This page provides specific testing information for the diagnosis of amebiasis at Public Health Ontario (PHO). The causative agent of amebiasis is the parasitic amoeba Entamoeba histolytica.

Updates
This new testing information webpage was published on October 15, 2025 and includes information that was previously found in: Entamoeba histolytica-Faeces-Para, Entamoeba histolytica- Differentiation, and Entamoeba histolytica -Abscess Liver.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 15 oct. 2025.

Testing Indications

Intestinal amebiasis: Microscopy is the current diagnostic method at PHO. However, it cannot distinguish Entamoeba histolytica from other morphologically identical but non-pathogenic Entamoeba species frequently colonizing the intestinal tract such as E. dispar, E. moshkovskii, and E. bangladeshi. Although some microscopic findings — like hypertrophy or hematophagy —are more commonly associated with E. histolytica, these findings are not exclusive and may occasionally (but rarely) appear in non-pathogenic species. Differentiation between these species in intestinal specimens requires additional confirmation by antigen or molecular testing. Note: Serology is NOT indicated for individuals with isolated intestinal amebiasis due to its limited accuracy in this situation.

Extraintestinal amebiasis (e.g., amebic liver abscess): Microscopy, antigen testing, and PCR can be performed directly on extraintestinal specimens in addition to histopathology and serology testing.

Acceptance/Rejection Criteria

Swabs other than fecal swabs are not accepted and will be cancelled.
Antigen and/or PCR test requests submitted in sodium acetate, acetic acid, and formalin (SAF) vials are not eligible for testing and will be cancelled.
Specimens submitted without relevant clinical or exposure information may be delayed or rejected.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Entamoeba histolytica microscopy	General Test Requisition	Intestinal specimens (e.g., feces, intestinal biopsy / aspirate / scraping, Entero-Test)	1.0 ml	SAF vial (see note 3)
Entamoeba histolytica microscopy	General Test Requisition	Extraintestinal specimens except blood (e.g. liver aspirate)	N/A	Empty sterile vial
Entamoeba histolytica antigen, molecular, or PCR	General Test Requisition	Intestinal or extraintestinal specimens except blood	N/A	Empty sterile vial or Cary-Blair vial

Submission and Collection Notes

Complete all fields of the requisition form.

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 submitting SAF-preserved intestinal specimens for microscopy: make sure that the specimen is mixed thoroughly with the SAF fluid as soon as collection occurs to preserve the specimen fully.

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

For intestinal specimens:

Avoid antacids or antimicrobials at least 2–3 weeks before collection as it can alter the intestinal microbiome.
Avoid laxatives/enemas (e.g., mineral/castor oil), nonabsorbable antidiarrheal preparations (e.g., bismuth), and kaolin at least 7–10 days before collection as it can affect the staining process.
Avoid contrast dyes (e.g., barium) at least 3 weeks before collection as it can affect the staining process.
Avoid contact between feces and urine or water during collection.

Storage and Transport

Place specimen container in a biohazard bag and properly seal the bag.

SAF specimens should be stored at room temperature. Other unpreserved or Cary-Blair specimens should be stored at 2—8°C and shipped to PHO on ice packs within 48 hours of collection. If unpreserved or Cary-Blair specimens might not be able to arrive at PHO within 48 hours of collection, they should be frozen at - 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)

Microscopy on intestinal specimens is performed daily from Monday to Friday at PHO’s laboratory - Toronto, Peterborough, Ottawa, and London locations . Turnaround time is up to 7 calendar days from receipt at those PHO locations.

Microscopy on extraintestinal specimens is performed daily from Monday to Friday at PHO’s laboratory, Toronto location. Turnaround time is up to 5 calendar days from receipt .

Antigen testing is performed twice weekly at PHO’s laboratory, Toronto location. Turnaround time is up to 7 calendar days from receipt at PHO’s laboratory.

Molecular testing is performed weekly at the National Reference Centre for Parasitology (NRCP) in Montreal. Turnaround time is up to 30 calendar days from receipt by NRCP.

Stat and Critical Specimens Testing

Priority testing for extraintestinal amebiasis 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

E. histolytica microscopy is performed at PHO using Hematoxylin or Giemsa staining along with diphasic sedimentation using formalin and ethyl acetate (FEA).

E. histolytica antigen testing is performed at PHO using the TECHLAB E. HISTOLYTICA II enzyme-linked immunosorbent assay (ELISA) test kit which detects E. histolytica specific galactose/N-acetylgalactosamine-binding lectin (Gal/GalNAc lectin, also called adhesin) antigens. These antigens are specific to E. histolytica trophozoites.¹

E. histolytica PCR testing is performed at the NRCP using a laboratory-developed assay targeting the small subunit (SSU) rDNA which can distinguish E. histolytica from E. dispar.

Performance and Limitations:
Microscopy has limited sensitivity (under 60% for intestinal infection; under 30% for extraintestinal infection) and poor specificity due to its inability to distinguish E. histolytica from nonpathogenic Entamoeba species such as E. dispar, E. moshkovskii, and E. bangladeshi.^1-2 Positive microscopy findings require antigen or molecular testing for species identification. Due to the intermittent shedding of organisms in feces, serial collection may be considered (e.g. two to three specimens over the course of two weeks) if the first fecal specimen result was initially negative and clinical suspicion remains high.

Antigen testing sensitivity is under 90% with specificity above 80%.^2-4 It can distinguish E. histolytica from nonpathogenic Entamoeba species. The assay does not detect the cyst form of E. histolytica and may miss asymptomatic cyst carriers or residual carriage following trophozoidal treatment. The assay has not been validated on extraintestinal specimens nor evaluated with E. moshkovskii or E. bangladeshi.

PCR performance at the NRCP is currently under investigation and is not yet validated on extraintestinal specimens. In other assays, PCR is usually estimated to have over 90% sensitivity and specificity². However, PCR performance is not well established in extraintestinal specimens and has not often been evaluated with E. moshkovskii or E. bangladeshi.

Algorithm

All unpreserved or Cary-Blair extraintestinal specimens will have both antigen and PCR testing performed routinely.
All unpreserved or Cary-Blair intestinal specimens will have the antigen testing performed ONLY if accompanied by a recent SAF-preserved positive microscopy result. If both SAF-preserved and unpreserved intestinal specimens were received at PHO, antigen testing will be automatically added if microscopy is positive. Otherwise, please indicate that specimen is known to be positive for E. histolytica/E. dispar/E. moshkovskii/E. bangladeshi on the requisition for antigen testing to be performed.

Interpretation

Microscopy:

Parasite microscopy	Interpretation
*E. histolytica/E. dispar/E. moshkovskii/E. bangladeshii*	Cannot distinguish between E. histolytica (pathogenic) from non-pathogenic Entamoeba species. If only an SAF-preserved specimen was received, please collect an unpreserved specimen as soon as possible for E. histolytica confirmation.
No parasites found	No evidence of E. histolytica organisms

Antigen testing:

Entamoeba histolytica antigen testing	Interpretation
Positive	Entamoeba histolytica trophozoite antigens detected.
Negative	Entamoeba histolytica trophozoite antigens NOT detected. Due to the limited test sensitivity, testing of additional specimens recommended if clinically indicated.
Invalid	Inconclusive result. Testing of additional specimens recommended if clinically indicated.

Note: Antigen testing is not yet validated on extraintestinal specimens at PHO. Results should be interpreted with caution.

PCR testing:

Entamoeba histolytica PCR	Interpretation
Positive	Entamoeba histolytica DNA detected.
Negative	Entamoeba histolytica DNA NOT detected.
Invalid	Inconclusive result. Testing of additional specimens recommended if clinically indicated.

Note: PCR testing is not yet validated on extraintestinal specimens at PHO. Results should be interpreted with caution.

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.

Positive specimens are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

References

Haque R et al. Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits. J Clin Microbiol. 1995 Oct; 33(10): 2558–2561.
Cooney J, Siakavellas SI, Chiodini PL, et al. Recent advances in the diagnosis and management of amoebiasis. Frontline Gastroenterology 2025;16:37-50.
Mirelman D et al. Comparison of use of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of Entamoeba histolytica and E. dispar. J Clin Microbiol. 1997 Sep; 35(9): 2405–2407
Roy S et al. Real-Time-PCR Assay for Diagnosis of Entamoeba histolytica Infection. J Clin Microbiol. 2005 May; 43(5): 2168–2172.