In-vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebas: Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri

The anticancer agent miltefosine and the antifungal drug voriconazole were tested in vitro against Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri. All three amebas are etiologic agents of chronic (Balamuthia, Acanthamoeba) or fulminant (Naegleria) encephalitides in humans and animals and, in the case of Acanthamoeba, amebic keratitis. Balamuthia exposed to <40 microm concentrations of miltefosine survived, while concentrations of >or=40 microM were generally amebacidal, with variation in sensitivity between strains. At amebastatic drug concentrations, recovery from drug effects could take as long as 2 weeks. Acanthamoeba spp. recovered from exposure to 40 microM, but not 80 microM miltefosin. Attempts to define more narrowly the minimal inhibitory (MIC) and minimal amebacidal concentrations (MAC) for Balamuthia and Acanthamoeba were difficult due to persistence of non-proliferating trophic amebas in the medium. For N. fowleri, 40 and 55 microM were the MIC and MAC, respectively, with no trophic amebas seen at the MAC. Voriconazole had little or no inhibitory effect on Balamuthia at concentrations up to 40 microg/ml, but had a strong inhibitory effect upon Acanthamoeba spp. and N. fowleri at all drug concentrations through 40 microg/ml. Following transfer to drug-free medium, Acanthamoeba polyphaga recovered within a period of 2 weeks; N. fowleri amebas recovered from exposure to 1 microg/ml, but not from higher concentrations. All testing was done on trophic amebas; drug sensitivities of cysts were not examined. Miltefosine and voriconazole are potentially useful drugs for treatment of free-living amebic infections, though sensitivities differ between genera, species, and strains.     

Balamuthia mandrillaris exhibits metalloprotease activities

Balamuthia mandrillaris is a recently identified protozoan pathogen that can cause fatal granulomatous encephalitis. However, the pathogenesis and pathophysiology of B. mandrillaris encephalitis remain unclear. Because proteases may play a role in the central nervous system (CNS) pathology, we used spectrophotometric, cytopathic and zymographic assays to assess protease activities of B. mandrillaris. Using two clinical isolates of B. mandrillaris (from human and baboon), we observed that B. mandrillaris exhibits protease activities. Zymographic assays revealed major protease bands of approximate molecular weights in the region of 40-50 kDa on sodium dodecyl sulfate-polyacrylamide gels using gelatin as substrate. The protease bands were inhibited with 1,10-phenanthroline, suggesting metallo-type proteases. The proteolytic activities were observed over a pH range of 5-11 with maximum activity at neutral pH and at 42 degrees C. Balamuthia mandrillaris proteases exhibit properties to degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue. This is shown by degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as other substrates such as casein and gelatin but not haemoglobin. However, these proteases exhibited a minimal role in B. mandrillaris-mediated host cell death in vitro using human brain microvascular endothelial cells (HBMECs). This was shown using broad-spectrum matrix metalloprotease inhibitors, GM 6001 and GM 1489, which had no effect on B. mandrillaris-mediated HBMEC cytotoxicity. This is the first demonstration that B. mandrillaris exhibits metalloproteases, which may play important role(s) in the ECM degradation and thus in CNS pathology.     

Identification of Naegleria fowleri and other Naegleria spp. (free-living amoebae) using cellulose acetate membrane electrophoresis of glucose phosphate isomerase

Abstract A simple isoenzyme cellulose acetate membrane electrophoresis method with respect to glucose phosphate isomerase (GPI) was developed for the differentiation of the human pathogenic free-living amoeba Naegleria fowleri from other Naegleria spp. A single GPI band was detected in all the species tested, the relative mobility of which could be used to identify N. fowleri . Of the other Naegleria spp., only N. italica and N. jadini shared a common GPI mobility. No intraspecies variation in GPI profile was detected, regardless of whether the strains were cultured in monoxenic or axenic media. The technique is proposed as a useful means of identifying N. fowleri soon after isolation from the environment.     

Balamuthia Mandrillaris, N. G., N. Sp., Agent of Amebic Meningoencephalitis In Humans and Other Animals

ABSTRACT. We recently reported the isolation of a leptomyxid ameba from the brain of a mandrill baboon that died of meningo-encephalitis. Based on light and electron microscopic studies, animal pathogenicity tests, and immunofluorescence patterns, we conclude that our isolate differs fundamentally from the other two amebas ( Leptomyxa and Gephyramoeba ) included in the Order Leptomyxida. We therefore created a new genus, Balamuthia , to accommodate our isolate and described it as Balamuthia mandrillaris to reflect the origin of the type species. Briefly, B. mandrillaris is a pathogenic ameba that causes amebic encephalitis in humans and animals. It has trophic and cyst stages in its life cycle, and is uninucleate with a large vesicular nucleus and a central nucleolus. Mature cysts have a tripartite wall consisting of an outer loose ectocyst, an inner endocyst and a middle mesocyst. Unlike Acanthamoeba and Naegleria , the other two amebas that cause amebic encephalitis in humans, Balamuthia will not grow on agar plates seeded with enteric bacteria. However, Balamuthia grows on a variety of mammalian cell cultures and kills mice following intranasal or intraperitoneal inoculation. Based on immunofluorescence testing, 35 cases of amebic encephalitis in humans and three in other animals have been identified worldwide as being caused by Balamuthia .