Acanthamoeba – Culture, PCR, and Microscopy

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 in vitro microscopy, culture, and PCR testing information for Acanthamoeba at Public Health Ontario (PHO). Acanthamoeba is a free-living ameba found in the environment worldwide which may cause amebic keratitis, chronic cutaneous infections, or granulomatous amebic encephalitis (GAE).

Updates

This page was updated to include the addition of collection instructions in the Special Instructions section and general information on method performance, limitations, and interpretation.

*Uncontrolled print copy. Valid only on day of print: 22 Oct 2026.

Testing Indications

Testing by culture and PCR is indicated for the diagnosis of individuals suspected of Acanthamoeba infection. In vitro microscopy of collected specimens has limited accuracy but may be available upon request. Although not available at PHO, direct in vivo confocal microscopy (IVCM) may be offered in some specialized clinics at the patient bedside by ophthalmologists with moderate to excellent accuracy.

Note: Acanthamoeba serology is not available at PHO.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Acanthamoeba	General Test Requisition	Body fluids or tissues (e.g., corneal scrapings, corneal biopsy, keratectomy tissue, brain biopsy, skin biopsy, cerebrospinal fluid) Used contact lens cases and fluid	1.0 ml (fluids) or 0.2 mg (tissues)	Sterile container

Submission and Collection Notes

See Special Instructions below for suggestions on specimen collection for corneal scrapings and biopsies.

Complete all fields of the requisition form.

If microscopy is requested beyond routine culture and PCR, clearly indicate “microscopy” on the requisition.

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.

Storage and Transport

Place specimen container in a biohazard bag and properly seal the bag. Specimens should be stored at room temperature and shipped to PHO as soon as possible within 24 hours of collection. All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Special Instructions

For corneal scrapings and biopsies, the following steps should be considered:

If in vivo confocal microscopy (IVCM) is planned to be performed, consider performing the examination prior to microbiological sampling to improve IVCM yield.
If possible, avoid administering topical dyes (e.g. fluorescein) or topical medications with high antimicrobial activity (e.g. preservatives, antibiotics, antifungals, antiparasitics) prior to sampling.
If the collection vial used does not already contain sterile normal (0.85%) saline, add a small amount (≤ 1.0 ml) to keep the sample hydrated during transport. Important: Do NOT use tap water.
Using a swab (polyester e.g. Dacron, rayon, or nylon preferred over cotton swabs), scalpel, needle, spatula, or punch tool, sample multiple suspected sites of infection including the base and the border of the corneal lesion(s).
Important: Do NOT leave the collection tool in the sample vial; promptly remove the tool from the vial once the liquid saline has been inoculated with the sample.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Acanthamoeba in vitro microscopy, culture, and PCR are performed daily from Monday to Friday at PHO’s laboratory-Toronto. Turnaround time for PCR is up to 5 calendar days from receipt. Turnaround time for culture is up to 14 calendar days from receipt. If requested, turnaround time for in vitro microscopy is up to 3 calendar days from receipt.

Test Methods

Acanthamoeba culture is performed routinely using a combined panel of four culture plates incubated at room temperature for up to 10 days: monoxenic E. coli in low-nutrient medium (0.2% peptone and 0.2% glucose) without salt, monoxenic E. coli in low-nutrient medium with salt (0.85% NaCl), monoxenic K. pneumoniae in low-nutrient medium without salt, and monoxenic K. pneumoniae in low-nutrient medium with salt.

Acanthamoeba PCR is performed routinely using a combined panel of two Acanthamoeba specific 18S rRNA gene targets, one from Riviere D et al. (2006), and one from Qvarnstrom Y et al. (2006).^1-2

If requested, Acanthamoeba in vitro microscopy is performed using direct wet mount and Giemsa staining.

Performance and Limitations:
Acanthamoeba culture sensitivity varies from 20-65%. Some Acanthamoeba species may grow preferentially at different temperatures, medium content, bacterial feed species, or salt concentrations.^3-5 Acanthamoeba culture may grow other non-pathogenic environmental amebae as well as Naegleria. Delays in specimen transport, as well as topical agents with antimicrobial effects in the collected sample, may lead to false negative culture results. ^6-12

Acanthamoeba PCR sensitivity ranges from 60-100%. There is no expected cross-reactivity with other non-pathogenic environmental amebae, Balamuthia, or Naegleria. Some uncommon Acanthamoeba species and genotypes may be missed by PCR, therefore culture is routinely performed in parallel. Topical agents with dyes or antimicrobial effects, as well as metallic or wooden tools left in the collected sample over time, may lead to false negative or invalid PCR results. ^6-12

If performed, in vitro microscopy sensitivity on clinical specimens is usually under 50%. Other non-pathogenic amebae, Balamuthia, and Naegleria may be difficult to differentiate by microscopy only. ^6-12

Algorithm

Culture and PCR testing are done routinely on all submitted samples. Due to its poor test accuracy, microscopy is only performed if specifically requested on the test requisition.

Interpretation

For Acanthamoeba culture results:

Acanthamoeba Culture	Interpretation
*Acanthamoeba* species found	Acanthamoeba organisms isolated. Acanthamoeba can be found in the environment. Results should be interpreted with caution if the specimen was not collected under sterile conditions.
Acanthamoeba species NOT found	Acanthamoeba organisms not isolated. PCR is a more sensitive test than culture, refer to PCR for confirmation.

For Acanthamoeba PCR results:

Acanthamoeba PCR	Interpretation
Detected	Acanthamoeba DNA present. Acanthamoeba can be found in the environment. Results should be interpreted with caution if the specimen was not collected under sterile conditions.
Indeterminate	Inconclusive result. An indeterminate PCR result may be due to a low level of target genetic material in the specimen, inadequate specimen content, or a non-specific signal. Please resubmit another specimen for testing if clinically indicated.
Not Detected	Acanthamoeba DNA not present. If only one specimen was submitted, repeat collection and testing may be advised if clinically indicated.
Invalid	No result available. 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.

For Acanthamoeba microscopy:

Parasite Microscopy	Interpretation
Parasite(s) found: Free-living amoebae	Free-living amebae can be found in the environment. Results should be interpreted with caution if the specimen was not collected under sterile conditions.
Parasite(s) NOT found	Refer to culture and PCR results for confirmation.

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.

References

Rivière D, Szczebara FM, Berjeaud JM, Frère J, Héchard Y. Development of a real-time PCR assay for quantification of Acanthamoeba trophozoites and cysts. J Microbiol Methods. 2006 Jan;64(1):78-83. doi: 10.1016/j.mimet.2005.04.008. Epub 2005 May 31. PMID: 15923051.
Qvarnstrom Y, Visvesvara GS, Sriram R, da Silva AJ. Multiplex real-time PCR assay for simultaneous detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri. J Clin Microbiol. 2006 Oct;44(10):3589-95. doi: 10.1128/JCM.00875-06. PMID: 17021087; PMCID: PMC1594764.
Bergmanson JP, Wang E, Gire AI, Osato MS. In vitro effects of medium tonicity, nutrient concentration, and free chlorine content on Acanthamoeba. Cont Lens Anterior Eye. 2011 Aug;34(4):164-8. doi: 10.1016/j.clae.2011.02.003. Epub 2011 Apr 22. PMID: 21514210.
de Moraes J, Alfieri SC. Growth, encystment and survival of Acanthamoeba castellanii grazing on different bacteria. FEMS Microbiol Ecol. 2008 Nov;66(2):221-9. doi: 10.1111/j.1574-6941.2008.00594.x. Epub 2008 Sep 22. PMID: 18811648.
Penland RL, Wilhelmus KR. Comparison of axenic and monoxenic media for isolation of Acanthamoeba. J Clin Microbiol. 1997 Apr;35(4):915-22. doi: 10.1128/jcm.35.4.915-922.1997. PMID: 9157153; PMCID: PMC229701.
Boggild AK, Martin DS, Lee TY, Yu B, Low DE. Laboratory diagnosis of amoebic keratitis: comparison of four diagnostic methods for different types of clinical specimens. J Clin Microbiol. 2009 May;47(5):1314-8. doi: 10.1128/JCM.00173-09. Epub 2009 Mar 25. PMID: 19321727; PMCID: PMC2681827.
Yera H, Ok V, Lee Koy Kuet F, Dahane N, Ariey F, Hasseine L, Delaunay P, Martiano D, Marty P, Bourges JL. PCR and culture for diagnosis of Acanthamoeba keratitis. Br J Ophthalmol. 2021 Sep;105(9):1302-1306. doi: 10.1136/bjophthalmol-2020-316730. Epub 2020 Oct 24. PMID: 33099504.
Khaksar S, Latifi A, Mohebali M, Atighehchian M, Moradian A, Asadigandomani H, Rezaian M, Anasori N, Koosha M, Pellegrino K, Izadi S, Goudarzi F, Kazemirad E, Soleimani M. Nested PCR as a superior diagnostic method for Acanthamoeba keratitis compared to conventional techniques. Sci Rep. 2026 Jan 27;16(1):3502. doi: 10.1038/s41598-025-32716-1. PMID: 41588033; PMCID: PMC12848294.
Hoffman JJ, Dart JKG, De SK, Carnt N, Cleary G, Hau S. Comparison of culture, confocal microscopy and PCR in routine hospital use for microbial keratitis diagnosis. Eye (Lond). 2022 Nov;36(11):2172-2178. doi: 10.1038/s41433-021-01812-7. Epub 2021 Nov 5. PMID: 34741122; PMCID: PMC9581916.
Tuft S, Bunce C, De S, Thomas J. Utility of investigation for suspected microbial keratitis: a diagnostic accuracy study. Eye (Lond). 2023 Feb;37(3):415-420. doi: 10.1038/s41433-022-01952-4. Epub 2022 Jan 31. PMID: 35102245; PMCID: PMC9905491.
Thompson PP, Kowalski RP, Shanks RM, Gordon YJ. Validation of real-time PCR for laboratory diagnosis of Acanthamoeba keratitis. J Clin Microbiol. 2008 Oct;46(10):3232-6. doi: 10.1128/JCM.00908-08. Epub 2008 Aug 13. PMID: 18701667; PMCID: PMC2566128.
Lau R, Cunanan M, Jackson J, Ali IK, Chong-Kit A, Gasgas J, Tian J, Ralevski F, Boggild AK. Reevaluation of an Acanthamoeba Molecular Diagnostic Algorithm following an Atypical Case of Amoebic Keratitis. J Clin Microbiol. 2015 Oct;53(10):3213-8. doi: 10.1128/JCM.01607-15. Epub 2015 Jul 22. PMID: 26202123; PMCID: PMC4572543.