Molecular, Immunological and Drug Sensitivity Studies of Pathogenic Free-living Amoebae

Balamuthia mandrillaris is a free-living amoeba that causes Balamuthia amoebic encephalitis, a fatal disease in humans and animals. Its ecology and risk to humans is largely unknown, although Balamuthia infections have been mostly associated with soil-related activities. Ecology studies are hampered by difficulty in isolating the amoeba by culture methods used for other free-living amoebae. In this study, a DNA extraction method and nested PCR was developed for rapid detection of B. mandrillaris from environmental samples, without needing primary culturing. More than 25% of soil samples were positive for B. mandrillaris, predominantly those from high temperature countries. Additionally, B. mandrillaris was frequently found in thermally polluted water, with almost 50% of samples positive. To facilitate the isolation of B. mandrillaris from environmental samples, immunomagnetic separation with B. mandrillaris antibodies was investigated. For this, poly- and monoclonal antibodies were produced and tested for specificity. Immunomagnetic separation isolated B. mandrillaris cysts but they were often contaminated with fungi, thus hindering further culture. In contrast, the technique was not suitable for the trophozoites due to toxicity and inability of the amoeba to survive the separation process. Treatment of B. mandrillaris infections is mainly with combinations of drugs of varying efficacy and with undesirable side-effects. Here, an improved in vitro drug sensitivity assay was developed for B. mandrillaris trophozoites and cysts. Diminazene aceturate was shown to be effective against trophozoites and cysts, with the minimum amoebacidal concentration of 7.8 μM and minimum cysticidal concentration of 62.5 μM. Naegleria fowleri is a thermophilic amoeboflagellate that causes primary amoebic meningoencephalitis, a fatal disease of the CNS. Conventional culture methods for its isolation from environmental samples may take several days, and thus a direct DNA extraction and rapid one-step nested PCR was utilised.