Identification of Antigenic Targets for Immunodetection of Balamuthia mandrillaris Infection

Abstract

The free-living amoeba Balamuthia mandrillaris causes granulomatous amoebic encephalitis (GAE) in humans. Rapid identification of balamuthiasis is critical for effective therapeutic intervention and case management. In the present study we identified target antigens for the development of a serological assay for B. mandrillaris infection. We demonstrated by silver staining that protein profiles for all eight isolates of B. mandrillaris, independent of human or animal origin or geographic origin, appeared to be similar except for some minor differences, indicating the molecular homogeneity of these strains. The profiles of all isolates, which ranged from 200 to 10 kDa, were similar, with a prominent protein visible around 30 kDa; all appeared considerably different from protein profiles of the control E6 cells and Acanthamoeba castellanii and Naegleria fowleri isolates. Western blot analysis with rabbit hyperimmune serum identified the major immunodominant antigens of 25, 50, 75, and 80 kDa; positive human sera reacted strongly with proteins around 25, 40, 50, and 75 kDa. Proteins around 40 kDa detected by human serum were not recognized by hyperimmune rabbit serum. None of the target proteins were detected by uninfected control sera. Reactivities of five patients' sera with 4 different isolates of B. mandrillaris (2 strains of human and 2 strains of animal origins) revealed that patients' sera reacted slightly differently with different B. mandrillaris isolates, although major proteins of approximately 25, 50, and 75 kDa were present in all extracts.

Serologic survey for exposure following fatal Balamuthia mandrillaris infection

“…Studies of patients with encephalitis have found that IFA and ELISA testing appear to be relatively specific for B. mandrillaris infection, suggesting that they are good markers for exposure (Kucerova et al 2011; Schuster et al 2001; Schuster et al 2008). Further, our findings confirm those from other studies showing that infections with other parasites, including infections with other amebae, do not yield false positives on B. mandrillaris antibody testing (Kiderlen et al 2009; Kiderlen et al 2010; Schuster et al 2001).…”

“…To grow amebae for antibody testing, we cultured B. mandrillaris (CDC:V619; isolated from the CSF of a GAE patient from Mississippi in 2010) on monolayers of monkey kidney (E6) cells as described before (Kucerova et al 2011) and harvested cultures after they cleared the monolayer by ingesting all of the tissue culture cells. We then chilled the flasks on ice for 2 to 5 minutes, shook them to dislodge the amebae, and washed them three times in Hanks’ balanced salt solution (HBSS; Gibco catalog no.…”

Assessment of blood–brain barrier penetration of miltefosine used to treat a fatal case of granulomatous amebic encephalitis possibly caused by an unusual Balamuthia mandrillaris strain

“…The HLF cell cultures were sub-cultured periodically and amebae were harvested and stored at −80°C. The amebae stored at −80°C were thawed and disrupted and the resulting protein antigens were subjected to SDS-PAGE (Kucerova et al 2011). Separated proteins were silver stained and compared with protein profiles of other isolates (Kucerova et al 2011).…”

“…The amebae stored at −80°C were thawed and disrupted and the resulting protein antigens were subjected to SDS-PAGE (Kucerova et al 2011). Separated proteins were silver stained and compared with protein profiles of other isolates (Kucerova et al 2011). Real-time PCR (Qvarnstrom et al 2006) was performed on DNA from brain tissue, CSF and cultured amebae, extracted using the DNeasy tissue and blood kit (QIAGEN).…”

“…Sections were either stained with H&E or reacted with rabbit anti- Balamuthia antiserum (IFA) as described previously (Visvesvara et al 1990) and examined using an Olympus BX-60 microscope. Anti- Acanthamoeba serum served as a negative control because anti- Balamuthia antibodies do not cross react with anti- Acanthamoeba antibodies (Kucerova et al 2011; Huang et al 1999; Kiderlen et al 2009). As with brain tissue, CSF was also cultured (Visvesvara et al 1990; Visvesvara et al 1993) and tested by real-time PCR.…”