Acanthamoeba keratitis is a painful and debilitating infection of the eye caused by an opportunistic amoeba that occurs ubiquitously in nature. From the discovery of this disease in 1973 until 1984, there were only 11 published cases. At present, an estimated 20 new cases are confirmed per month and the majority are associated with contact lens wear. Both awareness and incidence of the disease are thought to be increasing. The success of medical treatment is inconsistent and repeated surgical intervention often is required. There is considerable confusion over identification of clinical isolates. Cell morphology and affinity for specific antisera are the criteria used for identification, but these approaches often give ambiguous results. There is an urgent need for methods to detect small numbers of amebas and to identify the infectious organisms reliably. This project proposes the use of small subunit ribosomal RNA genes (18S rDNA) as a basis for development of genus-, species-, and strain- specific oligonucleotide probes suitable for laboratory and clinical identification of isolates. This one-year pilot project will focus on sequencing of 18S rDNA isolated from amoeba strains associated with human eye infections. Nuclear rDNA will be amplified by the polymerase chain reaction and then sequenced. Sequences will be determined using oligonucleotide primers, primer extension, and dideoxynucleotide chain termination. rDNA sequences then will be analyzed to determine the extent of sequence variability among strains and searched for subsets of sequences that would be suitable targets for probes with different levels of specificity (i.e. genus, species, or strain). Development of clinical probes would occur in subsequent projects if suitable sequence variability is discovered in this study.
| Byers, T J; Hugo, E R; Stewart, V J (1990) Genes of Acanthamoeba: DNA, RNA and protein sequences (a review). J Protozool 37:17S-25S |
