Isolation of Balamuthia mandrillaris-specific antibody fragments from a bacteriophage antibody display library.

Balamuthia mandrillaris is a protist pathogen that can cause encephalitis with a mortality rate of more than 95%. Early diagnosis followed by aggressive treatment is a pre-requisite for successful prognosis. Current methods for identifying this organism rely on culture and microscopy, antibody-based methods using animals, or involve the use of molecular tools that are expensive. Here, we describe the isolation of antibody fragments that can be used for the unequivocal identification of B. mandrillaris. B. mandrillaris-specific antibody fragments were isolated from a bacteriophage antibody display library. Individual clones were studied by enzyme-linked immunosorbent assay, and immunofluorescence. Four antibody clones showed specific binding to B. mandrillaris. The usefulness of phage antibody display technology as a diagnostic tool for isolating antibody fragments against B. mandrillaris antigens and studying their biological role(s) is discussed further.

Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts.

Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens.

Combined drug therapy in the management of granulomatous amoebic encephalitis due to Acanthamoeba spp., and Balamuthia mandrillaris.

Granulomatous amoebic encephalitis (GAE) is caused by two protist pathogens, Acanthamoeba spp., and Balamuthia mandrillaris. Although rare, it almost always results in death. In the present study, amoebae were treated with various combinations of clinically-approved drugs, targeting vital cellular receptors and biochemical pathways. The results revealed that among the seven different combinations tested, three proved highly effective against both Acanthamoeba castellanii as well as B. mandrillaris at a concentration of 100µM. These combinations included (i) prochlorperazine plus loperamide; (ii) prochlorperazine plus apomorphine; and (iii) procyclidine plus loperamide. In viability assays, none of the drug-treated amoebae emerged as viable trophozoites, suggesting irreversible amoebicidal effects. Four combinations of drugs tested showed varied potency against A. castellanii and B. mandrillaris at 100µM. The combination of haloperidol and loperamide was highly effective against A. castellanii at 100µM, but potent effects against B. mandrillaris were observed only at 250µM. Digoxin and amlodipine were effective against A. castellanii and B. mandrillaris at 100µM and 250µM, respectively. In contrast, the combination of apomorphine and haloperidol was effective against B. mandrillaris and A. castellanii at 100µM and 250µM, respectively. At 100µM, the combination of procyclidine and amiodarone was effective against neither A. castellanii nor B. mandrillaris. In this case, amoebicidal properties were observed at 750µM for A. castellanii, and 950µM for B. mandrillaris. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic encephalitis caused by A. castellanii and B. mandrillaris.