Naegleria fowleri (Primary Amebic Meningoencephalitis) – 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 PCR and microscopy testing information for Naegleria fowleri at Public Health Ontario (PHO). Naegleria fowleri is a free-living ameba found in warm water which may cause primary amebic meningoencephalitis (PAM). Infections mainly occur following intra-nasal exposure to warm water in the preceding 2 weeks. PAM manifestations may include rapidly progressive meningoencephalitis with non-specific brain imaging that may include leptomeningeal enhancements, hemorrhages, infarcts, vasogenic edema, or hydrocephalus. CSF samples may show high opening pressure and have hemorrhagic/xanthochromic and neutrophilic pleocytosis with high protein and low glucose levels.

*Uncontrolled print copy. Valid only on day of print: 19 Oct 2026.

Testing Indications

Testing by microscopy and PCR is indicated for the diagnosis of individuals suspected of N. fowleri infection. Microscopy should also be attempted locally using common approaches such as hematoxylin & eosin (H&E), periodic acid-Schiff (PAS), trichrome, Giemsa, or Wright-Giemsa stains. CSF should be examined locally within 30 minutes to 1 hour of collection for motile trophozoites by wet mount showing pseudopodal or flagellated motility. Acridine orange, and to a lesser extent Gram staining, may also assist in identifying amebic structures locally if methanol fixation is used instead of heat fixation.¹

Acute meningitis, including PAM, is a Disease of Public Health Significance (DOPHS) under the Health Protection and Promotion Act (HPPA). Additional testing and management recommendations are stated in the Ontario Ministry of Health Infectious Disease Protocol Appendix 1 for Disease: Meningitis.

Note: Naegleria serology and culture are not available at PHO.

Acceptance/Rejection Criteria

Testing for Naegleria requires prior approval by a PHO microbiologist or designate. Prior to submission — ideally after a physician has seen the patient but prior to specimen collection — contact PHO’s Laboratory Customer Service Centre at 416-235-6556/1-877-604-4567 or the After-Hours Emergency Duty Officer at 416-605-3113.

The following information is mandatory for testing to be approved:

History of present illness
Exposure history (e.g. occupational or recreational contact with water) and location
Past medical history
Treatment history
CSF results if performed including cell count, biochemical and microbiological testing
Imaging results

Specimen Collection and Handling

Specimen Requirements

Test Requested

Required Requisition(s)

Specimen Type

Minimum Volume

Collection Kit

Naegleria

General Test Requisition

Cerebrospinal fluid (CSF)

Tissue biopsy (e.g. brain biopsy, skin biopsy)

1.0 ml

0.2 g

Sterile container

Submission and Collection Notes

Contact PHO Laboratory Customer Service Centre to obtain pre-approval prior to submission.

Complete all fields of the requisition form.

Indicate “Urgent” and Naegleria clearly on the requisition.

For tissue biopsies: if the collection vial used does not already contain sterile normal (0.85%) saline or PBS, add a small amount (≤ 0.5 ml) to keep the sample hydrated during transport.

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.

Package urgent specimens separately from routine specimens using TDG compliant packaging and labelling.

Indicate “Urgent” clearly on the outside of the package to assist with intake.

Ship urgent specimens directly to PHO’s laboratory - Toronto: Directions to 661 University Shipping Dock for Clinical Samples.

Storage and Transport

Specimens should be stored at 2-8°C and shipped to PHO on ice packs urgently. All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

If approved, Naegleria PCR is referred out to the US Centers for Disease Control and Prevention (US CDC) upon request. Turnaround time for PCR is up to 7 calendar days from receipt at PHO.

Naegleria microscopy is performed daily at PHO’s Toronto site. Turnaround time for microscopy is up to 3 calendar days from receipt at PHO.

Test Methods

Naegleria PCR is performed at the US CDC using a Naegleria specific 18S rRNA gene target based on Qvarnstrom Y et al. (2006).² Naegleria microscopy is performed at PHO using direct wet mount and Giemsa staining.

Performance and Limitations:
The Naegleria PCR clinical sensitivity is not fully established due to the limited number of clinical cases, but the analytical performance suggests a limit of detection of ≤ 1 organism per 50 µl. Sensitivity may vary by genotype. There is no expected cross-reactivity with Acanthamoeba, Balamuthia, or other nonpathogenic Naegleria species.^2-3 Microscopy sensitivity on CSF specimens is variable, especially after prolonged storage and transport, therefore local microscopy should also be attempted if PAM is suspected.

Interpretation

For Naegleria PCR results:

Naegleria PCR	Interpretation
Detected	Naegleria fowleri DNA present. Requires urgent clinical assessment.
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	Naegleria fowleri DNA not present.
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 Naegleria microscopy:

Parasite Microscopy	Interpretation
Parasite(s) found: Free-living amebae	Free-living amebaecan be found in the environment. Results should be interpreted with caution if the specimen was not collected under sterile conditions.
Parasite(s) NOT found	Does not rule out infection. Refer to PCR for confirmatory results.

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.

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

References

Eger L, Pence MA. The Brief Case: A Case of Primary Amebic Meningoencephalitis (PAM) after Exposure at a Splash Pad. J Clin Microbiol. 2023 Jul 20;61(7):e0126922. doi: 10.1128/jcm.01269-22. Epub 2023 Jul 20. PMID: 37470480; PMCID: PMC10358179.
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.
Streby A, Mull BJ, Levy K, Hill VR. Comparison of real-time PCR methods for the detection of Naegleria fowleri in surface water and sediment. Parasitol Res. 2015 May;114(5):1739-46. doi: 10.1007/s00436-015-4359-5. Epub 2015 Feb 18. PMID: 25855343; PMCID: PMC4631115.