18S Ribosomal DNA Typing and Tracking of Acanthamoeba Species Isolates from Corneal Scrape Specimens, Contact Lenses, Lens Cases, and Home Water Supplies of Acanthamoeba Keratitis Patients in Hong Kong

Abstract

We examined partial 18S ribosomal DNA (Rns) sequences of Acanthamoeba isolates cultured in a study of microbial keratitis in Hong Kong. Sequence differences were sufficient to distinguish closely related strains and were used to examine links between strains obtained from corneal scrape specimens, contact lenses, lens cases, lens case solutions, and home water-supply faucets of patients with Acanthamoeba. We also looked for evidence of mixed infections. Identification of Acanthamoeba Rns genotypes was based on sequences of approximately 113 bp within the genus-specific amplicon ASA.S1. This permitted genotype identification by using nonaxenic cultures. Of 13 specimens obtained from corneal scrapes, contact lenses, lens cases, or lens case solutions, 12 were Rns genotype T4 and the remaining one was Rns genotype T3. The sequences of corneal scrape specimens of two patients also were the same as those obtained from their contact lenses or lens case specimens. A possible triple-strain infection was indicated by three different T4 sequences in cultures from one patient's lenses. Although faucet water used by patients to clean their lenses is a possible source of infections, specimens isolated from the faucets at two Acanthamoeba keratitis patients' homes differed from their corneal scrape or lens specimens. The overall results demonstrate the potential of this Rns region for tracking Acanthamoeba keratitis strains in infections and for distinguishing single-strain and closely related multiple-strain infections even when other microorganisms might be present with the cultured specimens. They also confirm the predominance of Rns genotype T4 strains in Acanthamoeba keratitis infections.

Morphological, physiological, molecular and phylogenetic characterization of new environmental isolates of Acanthamoeba spp. from the region of Bratislava, Slovakia

“…Therefore the methods for their identification in clinical and environmental samples are being developed in order to enable quick and exact detection of amoebae in various environments including the possibility to determine their pathogenicity. Several studies have reported the presence of Acanthamoeba in the environment (Červa 1971;Booton et al 2002;Ettinger et al 2003;Kilvington et al 2004;Tsvetkova et al 2004;Lorenzo-Morales et al 2006;Rezaeian et al 2008). This study confirms Table 1.…”

“…The dominance of this genotype was proven by the works of several authors (Schroeder et al 2001;Walochnik et al 2000b;Booton et al 2002Booton et al , 2005De Jonckheere 2003;Ledee et al 2003). The dominant genotype in GAE and other non-AK infections is T4 followed by T1, T10, T12 (Booton et al 2005), T5 (Barete et al 2007) and T2 (Walochnik et al 2008).…”

Acanthamoeba T4 and T15 genotypes associated with keratitis infections in Italy

“…The high proportion of Rns genotype T4 strains has been described as the main factor responsible for Acanthamoeba keratitis infections in many countries, i.e. in France (8/11, plus additional recent data) [24,25], in Greece (4/5) [12], in Hong Kong (12/13) [26], and in the UK and Iran (17/24) [8], thus providing further support to the evidence that the T4 genotype plays a prominent role in the epidemiology of AK human infections.…”

Acanthamoeba keratitis update—incidence, molecular epidemiology and new drugs for treatment

“…Approaches that have been useful include RFLP analysis of the entire mitochondrial genome, 12,20-25 riboprinting (described below) of 18S rDNA, 12,26 production of typespecific PCR amplimers, [27][28][29][30] analyses based on sequences of subgenic PCR amplimers [30][31][32] as well as the development of fluorescent oligonucleotide probes for in situ staining that are specific either for the genus Acanthamoeba or for genotype T4. 33 The recent introduction of a 'reverse dot-blot' technique holds promise for the eventual simultaneous detection and identification of all rDNA genotypes present in any specimen.…”