Frederick L. Schuster

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.

Efficacy of Novel Antimicrobials Against Clinical Isolates of Opportunistic Amebas

ABSTRACT We examined the effects of the macrolide antimicrobial agent azithromycin and phenothiazine compounds against clinical isolates of Acanthamoeba spp. and Balamuthia mandrillaris, opportunistic pathogens of human beings and other animals. Acanthamoeba growth was inhibited in vitro at 1,5, and 10 μg/ml of azithromycin, but not the macrolides, erythromycin, and clarithromycin. In experiments attempting to simulate in vivo conditions, azithromycin protected monolayers of rat glioma cells from destruction by Acanthamoeba at a concentration of 0.1 μg/ml, and delayed destruction at concentrations of 0.001 and 0.01 μg/ml. We concluded that the minimal inhibitory concentration of azithromycin was 0.1 μg/ml. Our results, however, suggested that the drug was amebastatic but not amebicidal, since ameba growth eventually resumed after drug removal. The phenothiazines (chlorpromazine, chlorprothixene, and triflupromazine) inhibited Acanthamoeba growth by 70‐90% at 5 and 10 μg/ml, but some of these compounds were toxic for rat glioma cells at 10 μg/ml. Azithromycin was not very effective against B. mandrillaris in an in vitro setting, but was amebastatic in tissue culture monolayers at concentrations of 0.1 μg/ml and higher. Balamuthia amebas showed in vitro sensitivity to phenothiazines. Ameba growth was inhibited 30‐45% at 5 μg/ml in vitro, but completely at 5 μg/ml in the rat glioma model. In spite of their potential as antiamebic drugs in Balamuthia infections, toxicity of phenothiazines limits their use in clinical settings.

Chemosensory Responses of Acanthamoeba castellanii: Visual Analysis of Random Movement and Responses to Chemical Signals

A visual assay slide chamber was used in conjunction with time‐lapse videomicroscopy to analyze chemotactic behavior of axenically grown Acanthamoeba castellanii. Data were collected and analyzed as vector scatter diagrams and cell tracks. Amebas responded to a variety of bacterial products or potential bacterial products by moving actively toward the attractant. Responses to the chemotactic peptide formyl‐methionyl‐leucyl‐phenylalanine (fMLP), lipopolysaccharide, and lipid A were statistically significant (P≤ 0.03), as was the response to fMLP benzylamide (P≤ 0.05). Significant responses to cyclic AMP, lipoteichoic acid, and N‐acetyl glucosamine were also found.

Animal Model Balamuthia Mandrillaris CNS Infection: Contrast and Comparison in Immunodeficient and Immunocompetent Mice: A Murine Model of “Granulomatous” Amebic Encephalitis

Balainnlhia mandrillaris and several species of Acanthamoeba are pathogenic “opportunistic” free-living amebas which cause granulomatous encephalitis (GAE) in humans and animals. The granulomatous component is negligible or absent, particularly in immunocompromised individuals. GAE is an “opportunistic” infection, usually seen in debilitated, malnourished individuals, in patients undergoing immunosuppressive therapy for organ transplants, and in Acquired Immunodeficiency Syndrome (AIDS). From around the world 156 cases of GAE have been reported from 1956 through October 1, 1995, 59 (26 in the USA) of them caused by B. mandrillaris, at least seven of them in AIDS patients. The present study was designed to compare and contrast the susceptibility of infection, the rate of infectivity and the histopathological changes within the CNS between the mutant, severe combined immunodeficient mice (SCID) infected with B. mandrillaris and the normal immunocompetent BALB-C mice. The SCID mouse is severely deficient in B and T lymphocytes, therefore lacking immunoglobulin and cell-mediated immunity. This mouse is also prone to develop early T cell lymphomas. One thousand amebic trophozoites and cysts of B. mandrillaris were intranasally and intraperitoneally inoculated in both strains of mice. Seventy percent of the intranasally inoculated SCID mice died due to CNS infection. Amebic trophozoites and cysts were found within CNS parenchyma without inflammatory response. Death occurred from 2 to 4 weeks after inoculation. By contrast only 10 percent of the intranasally inoculated BALB-C mice died with CNS infection showing the characteristic features of GAE. None of the intraperitoneally inoculated mice developed amebic infection. The SCID and BALB-C mice are logical models to study the structural alterations within the CNS of B. mandrillaris infection. This animal model recapitulates with excellent degree of fidelity several aspects of the pathogenesis and histopathological of free-living amebic infection in human beings.

Ultrastructure of mitosis in the amoeboflagellate Naegleria gruberi.

Naegleria gruberi is an amoeboflagellate found in soil; mitosis is restricted to the amoeboid phase of its life-cycle. Ultrastructural examination of mitotic stages has confirmed some aspects of karyokinesis reported in earlier light-microscopic studies and expanded on other features of nuclear division described in electron-microscopic studies of Naegleria. The nuclear envelope remained intact throughout division, the nucleolus persisted, and centrioles were not found. Chromosomes were indistinguishable at the ultrastructural level, nor was any evidence detected of sites of microtubular attachment to possible chromosomes. An interzonal body, formed during separation in two of the nucleolus, was not an invariable feature of mitosis. The same was true of the polar caps, which appeared to be little more than the ends of the mitotic spindle. It is suggested that, in line with comparable situations in other protists, expansion of the nuclear envelope is chiefly responsible for separation of the nucleus into two daughter nuclei.

Comparative Effects of Selected Azole Compounds on Trophic and Cystic Stages of Acanthamoeba polyphaga

ABSTRACT. A clinical isolate of Acunthamoeba polyphagu, associated with Acunthamoebu keratitis, was used for in vitro evaluation of antiamebic activity of selected azole compounds. Those antimicrobials tested included clotrimazole, bifonazole, fluconazole, itraconazole, and ketoconazole. The drugs were tested at varying concentrations on axenic trophic amebas and MgC12,‐induced cysts. On the basis of their effects on the two stages in the ameba life‐cycle, clotrimazole and bifonazole were the more promising of the azoles screened but, at concentrations tested, were amebastatic rather than amebicidal. Other strains of A. polyphaga and species of Acunthamoebu were used for comparing variation in response to these and other antimicrobial agents.

Virus-Like Particles in a Soil Ameboflagellate, Naegleria Gruberi

By virtue of the ecological niches they occupy, protozoa have ample opportunity to become infected with virus-like particles (VLPs). One of the better described and documented instances of such an infection is in Entamoeba histolytica, an endosymbiont of the human gut. Diamond and co-workers (2) have described several different VLPs in the nucleus and cytoplasm of Entamoeba, but the source of infection is not clear. These VLPs may have been acquired by the amebas during their residence in the intestinal lumen, an ideal habitat for numerous species of bacteria and viruses. Not resolved in this study is the relationship between the VLPs and the ability of Entamoeba to cause amebic dysentery. Does the presence of VLPs in the ameba serve as a means of enhancing virulence of the amebas? The VLPs apparently cause lysis of amebas under appropriate circumstances (2).