Genotypic identification of non-keratitis infections caused by the opportunistically pathogenic ameba genus Acanthamoeba.

The genus Acanthamoeba is an assemblage of approximately 20 named species of free-living amoebae inhabiting a wide range of ecological niches, and has a worldwide distribution. Individual Acanthamoeba have been isolated from soil, fresh and saltwater, humans, domestic and feral animals. In addition to natural habitats, Acanthamoeba has been identified as the cause of several diseases. It acts as an opportunistic pathogen in the painful and sight-threatening condition Acanthamoeba keratitis (AK) in otherwise healthy individuals. Acanthamoeba is also responsible for life threatening infections in patients with immune defense deficiencies (IDD). Acanthamoeba strains have also been associated with other infections, including those of the skin, eye, and brain (granulomatous amoebic encephalitis, GAE). These are generally observed in immunocompromised individuals. Variations in the pathogenicity of Acanthamoeba strains have been recognized in various laboratory studies, but the relevance of these results to human disease is unclear. Traditionally, Acanthamoeba has been classified using morphological markers such as cyst morphology and trophozoite size and shape. Originally, taxa of Acanthamoeba were categorized into 3 morphological groups based largely on the cyst morphology of the species. More recent molecular analyses, including several of our own studies using the nuclear and mitochondrial small subunit ribosomal RNA genes (ssu rDNA), supported the morphological group structure of the genus [1–4]. Our work has focused on the small subunit ribosomal RNA gene of the nucleus (Rns) and the equivalent gene from the mitochondrial genome (rns). Data collected over the last decade now allow us to quickly analyze a clinical or environmental sample using molecular methods and determine and classify the sequence genotype. Sequence similarities between isolates for the 2 genes have been examined to determine relationships between strains and to explore possible correlations to disease phenotypes. The molecular analyses suggest that 12–15 genotype groups exist, designated T1, T2, etc. [3,4]. The final number of genotypes is an active area of investigation, since it is dependent upon statistical criteria employed to distinguish types. Although the major morphological groups are supported by molecular analyses, a number of the named species have not been supported as unique monophyletic entities when examined by molecular methods. The primary focus of our earlier studies has been on the genotype of isolates obtained from AK infections. Thus far, nearly all AK cases examined in our lab (as well as in several other labs around the world) are due to infections involving a closely related group of strains sharing similar Rns and rns genotypes. While over 14 genotypic classes (sequence types) have been identified, AK-associated strains all are classified within a closely related group of genotypes. This group includes genotypes classified as T4, T3, and T11 (most AK strains are classified as T4). Further, these three genotype classes form a single monophyletic group, including a number of nominal species within the genus Acanthamoeba. In the present study we examine Acanthamoeba isolates that have been obtained from various non-AK human infections. The genotypes of these isolates have been determined using both nuclear and mitochondrial ribosomal RNA gene sequences. Phylogenetic comparison of the sequences indicates that, while many of the non-AK disease isolates are genotype T4 (the AK associated genotype), other more distantly related genotypes are also observed in non-AK infections.