Balamuthia mandrillaris (Balamuthiasis) – PCR and Microscopy

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Background
This page provides PCR and microscopy testing information for Balamuthia mandrillaris at Public Health Ontario (PHO). Balamuthia mandrillaris is a free-living ameba found in the environment worldwide which may cause chronic cutaneous infections or granulomatous amebic encephalitis (GAE). Infections mainly occur following traumatic skin exposure to soil or soil inhalation. Skin manifestations may include chronic painless erythematous or violaceous granulomatous plaques leading to tissue destruction over months to years. GAE manifestations may include subacute to chronic necrotic ring-enhancing brain lesions with vasogenic edema and hydrocephalus. CSF samples may have lymphocytic pleocytosis with high protein and low glucose levels.

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Testing Indications

Testing by PCR is indicated for the diagnosis of individuals suspected of Balamuthia infection. Microscopy has limited accuracy compared with PCR but may be available upon request.

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

Acceptance/Rejection Criteria

Testing for Balamuthia 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 soil) 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

Balamuthia

General Test Requisition

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

Cerebrospinal fluid (CSF)

0.2 g

1.0 ml

Sterile container

Submission and Collection Notes

Contact PHO Laboratory Customer Service Centre for pre-approval by PHO Microbiologist or designate prior to submission.

Complete all fields of the requisition form.

If microscopy is requested beyond routine PCR, clearly indicate “microscopy” 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.

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 as soon as possible within 24 hours of collection. 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, Balamuthia 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‘s laboratory-Toronto.

If requested, Balamuthia microscopy is performed daily from Monday to Friday at PHO. Turnaround time for microscopy is up to 3 calendar days from receipt at PHO‘s laboratory-Toronto.

Test Methods

Balamuthia PCR is performed at the US CDC using a Balamuthia specific 18S rRNA gene target from Qvarnstrom Y et al. (2006).¹

If requested, Balamuthia microscopy is performed using direct wet mount and Giemsa staining.

Performance and Limitations:
The Balamuthia 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 is highest for tissue biopsy specimens if amebic structures are seen by histopathology. Sensitivity is likely under 50% for CSF specimens, therefore negative CSF results cannot rule out infection. There is no expected cross-reactivity with Acanthamoeba or Naegleria.^1-2 If performed, microscopy sensitivity on CSF specimens is likely low.

Algorithm

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

Interpretation

For Balamuthia PCR results:

Balamuthia PCR	Interpretation
Detected	Balamuthia mandrillaris DNA present.
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	Balamuthia DNA not present. If performed on a CSF specimen, a negative result cannot rule out infection.
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 Balamuthia 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.

References

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.
Schuster FL, Yagi S, Gavali S, Michelson D, Raghavan R, Blomquist I, Glastonbury C, Bollen AW, Scharnhorst D, Reed SL, Kuriyama S, Visvesvara GS, Glaser CA. Under the radar: balamuthia amebic encephalitis. Clin Infect Dis. 2009 Apr 1;48(7):879-87. doi: 10.1086/597260. PMID: 19236272.