Diagnosis & Detection
Initial testing: CSF typically demonstrates a predominantly lymphocytic pleocytosis with typically fewer than 500 cells/mm3. CSF glucose concentration may be normal or low and CSF protein concentration is elevated. Balamuthia organisms are rarely seen in the CSF. In the U.S. case series of 109 patients, only one patient was reported to have had amebas visualized on a wet mount of the CSF. This is in contrast to primary amebic meningoencephalitis caused by Naegleria fowleri where amebas are often visualized in the CSF of affected patients.
Balamuthia Case definition: In 2011, the Council of State and Territorial Epidemiologists (CSTE) established a standard case definition for Balamuthia infections. Laboratory-confirmed Balamuthia infection is defined as the detection of Balamuthia as:
- Organisms in CSF, biopsy, or tissue specimens, or
- Nucleic acid in CSF, biopsy, or tissue specimens, or
- Antigen in CSF, biopsy, or tissue specimens.
Tests available: Diagnostic testing is not widely available for Balamuthia infection. Clinicians who suspect Balamuthia infection should contact their state health department and/or CDC (24/7 Emergency Operations Center—770-488-7100). CDC can assist with diagnosis and provide treatment recommendations. Telediagnosis can be arranged at CDC by emailing photos through DPDx, CDC’s telediagnosis tool. Instructions for submitting photos through DPDx are available at the DPDx Contact Us page.
CSF: Balamuthia trophozoites and/or cysts are rarely seen in the CSF. Every effort should be made to obtain brain tissue in order to make the diagnosis of Balamuthia GAE. If Balamuthia is identified in the CSF, the diagnosis of GAE should be subsequently confirmed with PCR or immunohistochemical or IIF assays of the CSF because host cells can be mistaken for Balamuthia. Note that a negative test on CSF does not rule out Balamuthia infection because the organism is not commonly present in the CSF.
Tissue: The diagnosis of Balamuthia infection can be made by microscopic examination of tissue sections from biopsy specimens (skin lesions or brain tissue) stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS) which might demonstrate trophozoites and/or cysts with morphology typical of Balamuthia (Figures A–D).
The cysts of Balamuthia mandrillaris are 6–30 µm in diameter (Figure A and B).Under a light microscope, the cysts appear to have two walls: a wrinkled fibrous outer wall (exocyst) and an inner wall (endocyst) that may be hexagonal, spherical, star-shaped or polygonal. Refractile granules might be observed below the inner wall. Pores are not evident in the wall complex. Cysts usually contain only one nucleus but occasionally have two nuclei.
A, B: Cysts of B. mandrillaris in brain tissue, stained with H&E. Images courtesy of the University of Kentucky Hospital, Lexington, Kentucky.
Trophozoites of Balamuthia mandrillaris are pleomorphic and measure approximately 12–60 µm (Figure C and D). They often produce long, slender pseudopodia. Trophozoites are usually uninucleate but binucleate forms are sometimes seen. The nucleus contains a large, centrally-located nucleolus but two or three nucleoli have been seen, especially in infected tissues; when present, multiple nucleoli distinguish B. mandrillaris from Acanthamoeba spp. There is no flagellate trophozoite stage as in Naegleria spp.
C, D: Trophozoites of B. mandrillaris in brain tissue, stained with H&E.
Biopsies of skin lesions demonstrate granulomatous inflammation with infiltrating giant cells, lymphocytes, plasma cells, and eosinophils; Balamuthia cysts or trophozoites can often be seen in tissue sections . Although most lesions are found in the skin or the brain, granulomas containing amebas have also been found in other organs including the lungs and kidneys.
An increasing number of PCR-based techniques (conventional and real-time PCR) have been described for detection and identification of free-living amebae in clinical samples, but are only available in selected reference laboratories. A real-time PCR was developed at CDC for simultaneous identification and differentiation of Balamuthia mandrillaris, Naegleria fowleri, and Acanthamoeba species in clinical samples. This assay uses distinct primers and TaqMan probes for the simultaneous identification of these three amebas. Unlike Acanthamoeba, Balamuthia cannot be grown on agar plates coated with bacteria but requires mammalian cell cultures (such as monkey kidney [E6] or human lung fibroblasts) for laboratory cultivation.
Detecting Balamuthia mandrillaris antigen involves immunohistochemical (IHC), or indirect immunofluorescence (IIF) staining, which use rabbit anti-ameba sera that detects Balamuthia mandrillaris followed by microscopic examination to identify Balamuthia mandrillaris in tissue, or CSF (Figure E).
E: Indirect Immunofluorescence (IIF) assay for Balamuthia mandrillaris
Antibodies have been demonstrated in healthy persons and patients with Balamuthia infection. Serology using an indirect immunofluorescent antibody (IFA) test for Balamuthia is available at CDC, but is not used as a routine diagnostic test.
Diagnostic Tests Offered by CDC
Below are instructions for submitting specimens to CDC for free-living ameba testing. Please see the CDC Infectious Diseases Laboratory Test Directory for additional information.
Specimens can be sent to CDC for diagnostic assistance. If possible, please send the following specimens:
- Fresh, unfixed tissue or CSF
- Frozen samples, fresh unpreserved samples, samples reserved in ethanol or commercial fixatives designed to be compatible with molecular testing are suitable for PCR. If the specimen is preserved in formalin, CDC will still accept the specimen but note that formalin-fixed samples are not the preferred sample-type for molecular testing (e.g., PCR or real-time PCR).
Fresh, unfixed specimens (i.e., tissue and CSF) should be sent at ambient temperature by overnight priority mail. Fresh tissue sample should be sent in small volume (e.g., 0.5 mL) of equal volume mixture of sterile water and physiological saline to keep it moist. Please ship these specimens separately from other chilled or frozen samples being shipped.
Please send previously frozen tissue or CSF by overnight priority mail on ice packs (DO NOT ship on dry ice). Formalin-fixed (wet) tissues and formalin-fixed, paraffin-embedded (FFPE) tissue blocks should be shipped at ambient temperature.
Care should be taken to pack glass slides securely, as they can be damaged in shipment if not packed in a crush-proof container.
For fresh, unfixed specimens, frozen tissues, CSF, or H&E-stained or unstained slides only, please use the CDC specimen submission form pdf icon[PDF – 2 pages] and send to the address below. Be sure to add the pertinent travel history as well as relevant clinical history and any results from previous infectious disease testing. Please arrange Monday–Friday delivery only. Packages cannot be accepted on weekends or federal holidays.
CDC Shipping address:
Attn: Unit 53
1600 Clifton Road, NE
Atlanta, GA 30333
See Specimen Submission Guidelines for Pathologic Evaluation of CNS Infections for more information.
- For submission of Formalin-fixed (wet) tissue or formalin-fixed paraffin-embedded (FFPE) tissue blocks
Telediagnosis can be arranged at CDC by emailing photos through DPDx, CDC’s telediagnosis tool. Instructions for submitting photos through DPDx are available at the DPDx Contact Us page.
Please contact the DPDx Contact Us page before collecting samples if further information or guidance is required.