Diagnosis & Detection

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 tests 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)⁶ 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 flagellated trophozoite stage as in Naegleria spp. ^3,6,7.

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 ⁸. 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 ⁹.

E: Indirect Immunofluorescence (IIF) assay for Balamuthia mandrillaris.

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 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 amebae ¹⁰. Culture may be used to amplify the number of organisms for PCR testing, but is not used alone for diagnostic testing. 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 ⁷.

Detecting Balamuthia mandrillaris antigen involves immunohistochemical staining techniques, such as indirect immunofluorescence (IIF) staining and immune alkaline phosphatase staining (IHC), which use an antibody specific for Balamuthia mandrillaris followed by microscopic examination to identify Balamuthia mandrillaris in tissue, culture, or CSF.

Immunohistochemical (IHC) techniques employing rabbit anti-ameba sera ^{3, 11} with subsequent microscopic examination can identify Balamuthia in tissue, culture, or CSF. Three IHC methods are available: indirect immunofluorescence (IIF) staining (Figure E), immune peroxidase staining, or immune alkaline phosphatase staining.

Antibodies have been demonstrated in healthy persons and patients with Balamuthia infection ^3,12. Serology using an immunofluorescent antibody test for Balamuthia is available at CDC as a research technique, as it has not been evaluated for use as a routine diagnostic procedure.