Advances in Free‐living Amebae Research 2003: Workshop Summary

Presentations on free-living amebae causing granulomatous amebic encephalitis, amebic keratitis and primary amebic meningoencephalitis presented at The VIII International Workshops on Opportunistic Protists held in Hilo, Hawaii focused on ecological studies, genotyping, molecular biology, therapy, and host-parasite interactions. Papers and posters presented at the meeting are summarized below. Free-living amebae belonging to the genera Acanthamoeba, Balamuthia, and Naegleria can cause fatal CNS infections in human. Acanthamoeba are ubiquitous in nature and are one of the most commonly isolated amebae from the environment. Acanthamoeba has been isolated from fresh and marine water habitats, which can be used for recreational purposes. Munson and Paget (Washington College and University of Hull) reported on the impact of pollutants on the isolation rates and pathogen distribution of pathogenic Acanthamoeba spp. in the North Sea coastal environments. Six sites along the North Sea coast (United Kingdom) were sampled. Acanthamoeba spp. were isolated from 34 of 41 sediment samples. The results of their study demonstrate that species diversity is reduced in polluted environments and the majority of pathogens are isolated from ‘‘clean’’ water. Identification of Acanthamoeba at the genus level is relatively easy due to the presence of surface projections termed acanthapodia. Identification at the species level is problematic because many species share antigenic determinants. Fuerst et al. (Ohio State) have devised a molecular classification scheme using both nuclear and mitochondrial ribosomal gene sequences (www.biosci-ohiostate/;tbyers/ byers.htm). Using these techniques, approximately12 to15 genotypic classes designated T1, T2 to T15 have been identified. Less than 50 % of environmental isolates fall into the T4 genotype, whereas over 93 percent of amebic keratitis isolates fall into the T4 gentoytpe. Nonkeratitis human infection isolates fall into multiple genotypes including T1, T4, T10, and T12. Marciano-Cabral et al. (Medical College of Virginia) reported on the coculture of a clinical isolate of Acanthamoeba sp. with peritoneal macrophages from B6C3F1 mice. The clinical isolate from an immune suppressed patient who had undergone renal transplant surgery was found to harbor bacteria by transmission electron microscopy. The bacteria were nonculturable on standard bacteriological media. The interaction of the isolate with peritoneal macrophages demonstrated that the macrophages were capable of ingesting amebae and producing high levels of TNFa mRNA. The presence of bacteria within the amebae may exacerbate CNS and peripheral infections. Schuster and colleagues (California Dept. Health Services) investigated the amebostatic and amebicidal activity of a drug, myristamidopropyl dimethylamine (Aldox) for corneal isolates of Acanthamoeba from amebic keratitis patients. Their studies demonstrated that the drug was amebicidal in vitro at 10 to 20 lg/ml. The concentration effective against amebae was not toxic to normal corneal epithelial cells. Balamuthia mandrillaris, a member of the family Acanthamoebidae, has been isolated from humans and from zoo animals that have died from granulomatous amoebic encephalitis. Until the present time the ameba had not been isolated from the environment. Failure to isolate amebae from environmental sources questioned the free-living status of the ameba. Dunnebacke et al. (California Dept. Health Services) isolated an ameba morphologically similar to Balamuthia from a soil sample in the environment of an encephalitis victim and from a distal area. Molecular techniques developed by Booton et al. (Ohio State) were used to compare isolates from human tissue and soil. The amebae found in the environment and the human isolates were found to be similar indicating that Balamuthia is a free-living ameba capable of causing fatal infections in humans. In an effort to develop methods for more rapid identification of B. mandrillaris in the environment and in clinical samples, Booton et al. (Ohio State) have sequenced the nuclear 18S and mitochondrial 16S small subunit rRNA genes in a number of Balamuthia isolates. Using information based on the mitochondrial ssu rDNA, this group developed a Balamuthia-specific primer set that exclusively identifies the target region using DNA extracted from Balamuthia samples. This technique allowed for the identification of Balamuthia in environmental and clinical samples. The genus Naegleria is composed of several species of amoeboflagellates found in fresh water lakes and ponds and a number of recreational water sites such as thermal spas and swimming pools. Only one species, N. fowleri, has been associated with fatal human CNS infection. The distribution of N. fowleri in the environment is not well documented. A survey of 510 thermal waters in Japan was undertaken and reported by Endo et al. (National Institute of Infectious Dis. Japan) in order to determine the prevalence of thermotolerant N. fowleri. Forty-five percent of the water samples contained a variety of amebae. However, N. fowleri was not isolated but N. australiensis, a species pathogenic for mice was isolated from the water samples. Although N. australiensis is pathogenic for mice it has not been isolated from humans with amebic meningoencephalitis. Two very closely related species, pathogenic N. fowleri and nonpathogenic N. lovaniensis, cannot be distinguished by surface antigens. Zhou et al. (Center for Disease Control) discussed molecular studies using mitochondrial ssu rRNA gene sequences, which should be helpful in the identification of the two species. In conclusion, although free-living amebae are ubiquitous in the environment their identification at the species level is difficult. The majority of presentations focused on molecular methods of identification of the free-living amebae.