Extraintestinal Entamoeba histolytica (Amebic Abscess) – Microscopy, PCR, and Antibody

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

For testing of enteric specimens in the context of intestinal amebiasis, refer to the following PHO webpage: Enteric Protozoa (Cryptosporidium, Cyclospora, Dientamoeba, Entamoeba, and Giardia) - PCR.

Updates
This page has been revised to focus exclusively on extraintestinal amebiasis testing. Information around intestinal amebiasis testing should be referred to the enteric protozoa PCR webpage: Enteric Protozoa (Cryptosporidium, Cyclospora, Dientamoeba, Entamoeba, and Giardia) - PCR.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 18 déc. 2025.

Testing Indications

Microscopy, PCR, and serology may be indicated for the diagnosis of individuals suspected of extraintestinal amebiasis (e.g. amebic liver, brain, lung, or other abscesses; or perineal ulcerative lesions), in addition to histopathology.

Note: PCR testing is not yet validated at PHO on extraintestinal specimens; result must be interpreted with caution.

Acceptance/Rejection Criteria

Testing will be performed only when at least one of the following criteria is documented on the requisition:

Symptoms compatible with extraintestinal amebiasis (e.g. liver, brain, lung, or other abscesses; or perineal ulcerative lesions)
Close contact of an infected person (e.g. household or outbreak cluster)

Requests for patients with suspected intestinal amebiasis only (without extraintestinal invasion) will be cancelled.
PCR test requests submitted in sodium acetate, acetic acid, and formalin (SAF) vials are not eligible for testing and will be rejected.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit

Extraintestinal Entamoeba histolytica - PCR	General Test Requisition	Sterile body fluid or tissue specimens (e.g. abscess aspirate, skin biopsy)	1.0 ml (fluid)	Sterile empty vial
Entamoeba histolytica serology	General Test Requisition	Serum, or Blood, clotted	1.0 ml or 5.0 ml	Serum separator tube (SST), or Plain or pro-coagulation tube

Submission and Collection Notes

Complete all fields of the requisition form.

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

Symptoms compatible with extraintestinal amebiasis (e.g. liver, brain, lung, or other abscesses; perineal ulcerative lesions)
Close contact of an infected person (e.g. household 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.

Timing of Specimen Collection

For serology: results may be negative within the first 7 to 14 days after symptom onset; repeat testing ≥ 14 days after symptom onset may be indicated if extraintestinal amebiasis is strongly suspected.

Limitations

For serology: Grossly haemolysed, lipemic, contaminated specimens, and specimens containing anti-coagulant are unsuitable for testing.

Storage and Transport

Place specimen container in a biohazard bag and properly seal the bag. Centrifuge tube if using SST for serum specimens.

Unpreserved specimens and serum specimens should be stored at 2-8°C and shipped to PHO on ice packs within 48 hours of collection; if delayed beyond 48 hours of collection, store unpreserved specimens frozen at -20°C. 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 and routine PCR testing are performed daily from Monday to Friday at PHO’s Toronto site. Turnaround time is up to 5 calendar days from receipt.

Confirmatory PCR testing and serology are forwarded to the National Reference Centre for Parasitology (NRCP) in Montreal. Turnaround time is up to 42 calendar days from receipt at PHO.

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

Microscopy is performed at PHO using Giemsa staining along with standard sedimentation. Initial PCR testing is performed at PHO using the commercial Seegene Allplex GI-Parasite PCR multiplex assay. Confirmatory 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. Serology is performed at the NRCP using an enzyme-linked immunosorbent assay (ELISA) based on antibody capture using antigens of the E. histolytica strain NIH-200.

Performance and Limitations:
Microscopy has a sensitivity of under 30% for extraintestinal amebic infection. It is unable to distinguish E. histolytica from nonpathogenic Entamoeba species such as E. dispar, E. moshkovskii, and E. bangladeshi.^1-2

PCR performance at PHO and the NRCP is currently under investigation and is not yet validated on extraintestinal specimens. In other assays, PCR sensitivity ranges from 87-95% and specificity is usually 98% or higher. The assay has not been evaluated with E. moshkovskii or E. bangladeshi. ²

For serology, the NRCP reports a sensitivity above 95% for extraintestinal amebiasis and a specificity of 92%. With other assays, E. histolytica serology has a reported sensitivity of around 95% for extraintestinal amebiasis and a specificity above 90%.3 However, antibody titres may take 7 to 14 days to develop and may persist for years after resolution of the infection. Therefore, a negative result in acute illness does not rule out infection, and a single positive result cannot distinguish current from resolved or past infection. Note: Serology is poorly sensitive for localized or invasive intestinal amebiasis, therefore stool testing is preferrable over serology for individuals without extraintestinal manifestations.

Algorithm

Until PCR testing is validated at PHO, all PCR requests on extraintestinal specimens will be tested in parallel at PHO and the NRCP. At PHO, only E. histolytica will be reported on the multiplex PCR assay; other targets will not be reported on extraintestinal specimens.

Interpretation

Microscopy:

Parasite Microscopy	Interpretation
Parasite(s) found: *E. histolytica*	The organism stage(s) will be reported. Cannot distinguish E. histolytica (pathogenic) from non-pathogenic Entamoeba species, however the presence of the organism in extraintestinal sites usually suggests pathogenicity.
No parasites found	No evidence of E. histolytica organisms. Due to limited sensitivity, testing of additional specimens by PCR may be considered if clinically indicated. If not already requested, serology may be advised if clinically indicated.

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.

Entamoeba histolytica serology by EIA:

Optical Density (OD) Sample Value	Result	Interpretation
≥ 1.20	High Positive	E. histolytica antibodies detected. Does not distinguish current from resolved or past infection. Clinical correlation required.
0.45-1.19	Low Positive	E. histolytica antibodies detected. Does not distinguish current from resolved or past infection. Clinical correlation required.
0.35-0.44	Equivocal	Inconclusive results. Repeat collection if clinically indicated.
< 0.35	Negative	E. histolytica antibodies NOT detected. False negative results may occur in the first 2 weeks of symptom onset. Repeat collection if clinically indicated.

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.
The NRCP reports a sensitivity above 95% for extraintestinal amebiasis and a specificity of 92%. With other assays, E. histolytica serology has a reported sensitivity of around 95% for extraintestinal amebiasis and specificity above 90%.1 However, antibody titres may take 7 to 14 days to develop and may persist for years after resolution of the infection. Therefore, a negative result in acute illness does not rule out infection, and a single positive result cannot distinguish current from resolved or past infection. Note: Serology is poorly sensitive (≤ 70%) for invasive intestinal amebiasis, therefore stool testing is preferrable over serology for individuals without extraintestinal manifestations.