Laboratory investigation of Acanthamoeba lugdunensis from patients with keratitis.

PURPOSE

Species of four Acanthamoeba isolates (KA/E2, KA/E12, KA/E15, and KA/E16) from the cornea of patients with keratitis were identified and their molecular characteristics compared with those of other strains.

METHODS

Morphologic features of amebic cysts were evaluated with a microscope with differential interference contrast (DIC) optics. Restriction fragment length polymorphisms (RFLP) of mitochondrial DNA (mtDNA), riboprinting of small subunit ribosomal RNA gene (18S rDNA), and DNA sequences of 18S rDNA were analyzed. mtDNA and PCR-amplified 18S rDNA of the ocular isolates were digested with restriction enzymes, and the restriction patterns were compared with those of reference strains purchased from American Type Culture Collection (ATCC, Manassas, VA). PCR products of 18S rDNA were cloned and subjected to sequencing. The complete sequence of approximately 2300 bp obtained from the isolates and reference strains were compared with each other and those registered in GenBank.

RESULTS

Three ocular isolates (KA/E2, KA/E12, and KA/E16) of Acanthamoeba revealed the identical mtDNA RFLPs and riboprint patterns with Acanthamoeba L3a, the type strain of A. lugdunensis. The other isolate (KA/E15) had riboprint patterns very similar to A. lugdunensis L3a but quite different mtDNA RFLP patterns from those of all the other strains. A dendrogram based on the riboprint data showed that three ocular isolates were identified as A. lugdunensis and the other isolate was very closely related to this species. Identification of the isolates as A. lugdunensis was confirmed by 18S rDNA sequence analysis. The sequence differences of the four isolates from A. lugdunensis L3a was 0.1% to 0.4% (3 to 8/2284 bp) and 1.2% to 1.5% from A. castellanii Castellani.

CONCLUSIONS

This is the first report of Acanthamoeba keratitis in Korea caused by A. lugdunensis, which was originally isolated from a freshwater pool in France. Riboprinting can be used as a simple and rapid tool for putative identification of unknown Acanthamoeba ocular isolates.