Characterization of a new pathogenic Acanthamoeba Species, A. byersi n. sp., isolated from a human with fatal amoebic encephalitis

Abstract

Acanthamoeba spp. are free-living amoebae that are ubiquitous in natural environments. They can cause cutaneous, nasopharyngeal, and disseminated infection, leading to granulomatous amebic encephalitis (GAE) in immunocompromised individuals. In addition, they can cause amoebic keratitis in contact lens wearers. Acanthamoeba GAE is almost always fatal because of difficulty and delay in diagnosis and lack of optimal antimicrobial therapy. Here, we report the description of an unusual strain isolated from skin and brain of a GAE patient. The amoebae displayed large trophozoites and star-shaped cysts, characteristics for acanthamoebas belonging to morphology Group 1. However, its unique morphology and growth characteristics differentiated this new strain from other Group 1 species. DNA sequence analysis, secondary structure prediction, and phylogenetic analysis of the 18S rRNA gene confirmed that this new strain belonged to Group 1, but that it was distinct from the other sequence types within that group. Thus, we hereby propose the establishment of a new species, Acanthamoeba byersi n. sp. as well as a new sequence type, T18, for this new strain. To our knowledge, this is the first report of a Group 1 Acanthamoeba that is indisputably pathogenic in humans.

Keywords: 18S rRNA gene; Group 1 Acanthamoeba; human isolate, nuclear small subunit ribosomal RNA gene; ribosomal secondary structure; sequence type T18.

Figure 1

Acanthamoeba CDC:V621 trophozoites exhibiting varying sizes measuring from 50 to 110 μm. The trophozoites feature single (A–C) or double (D) nuclei or donut‐shaped nucleus (A, B). All trophozoites feature acanthopodia that are characteristically blunt at the tip, contractile vacuole, food vacuole, and a contractile vacuole. The large trophozoite is binucleate, indicating that it has completed karyokinesis and is awaiting cytokinesis. Bar = 10 μm.

Figure 2

Cysts of CDC:V621 featuring the unique morphology of a nearly round ectocyst and stellate endocyst with five to eight arms or rays at different planes (A, B). Pores are present at the junction points of the ecto‐ and endocysts. The pores are plugged with the operculum (arrows), which is removed at the time of excystation (arrowhead in C). Bar = 10 μm.

Figure 3

A predicted secondary structure for clone 4 of the 18S rRNA gene of CDC:V621 with areas of micro‐heterogeneity among the 10 clones indicated by arrows and the clone where the variation occurred designated with the clone number. The long inserted stem, E23‐13‐2, is outlined by a thick three‐sided box. Insertions are indicated by an arrow that does not point to a specific base. Deletions are indicated by an arrow pointing away from the specific base that has been deleted. Transitions and transversions are indicated where a base at the shaft end of the arrow is different from the base at the arrowhead point.

Figure 4

Phylogram based on 18S rRNA gene sequences from Acanthamoeba Group 1 strains. A representative from Group 2 (i.e., the type strain of A. castellanii) was added as the outgroup. All 10 sequences obtained from CDC:V621 formed a single clade, separate from other Acanthamoeba sequences. The phylogenetic tree was created with the quartet puzzling maximum likelihood method using the program TREE‐PUZZLE. The scale bar estimates evolutionary distance (nucleotide substitutions per position).