Acanthamoeba griffini. Molecular characterization of a new corneal pathogen

Purpose. Acanthamoeba was isolated from the cornea of a soft contact lense wearer who had keratitis. The protozoan was also isolated from the contact lens storage case and the domestic water supply used to clean the case. Using morphologic features, all three isolates were identified tentatively as A. griffini, a species not previously associated with keratitis. Complete small subunit ribosomal RNA gene (18S rDNA) sequence analysis was used to characterize further the three isolates. Methods. 18S rDNA was polymerase chain reaction-amplified from whole cell DNA derived from amoebal lysates. The genes were cloned and sequenced. Complete sequences of approximately 2800 base pairs were obtained from each culture and compared with those stored in a data base of homologous Acanthamoeba sequences. Results. The isolates were unequivocally identified as A. griffini both by comparison of the gene sequence available for the type strain of the species and the presence of a unique group I intron located within the small subunit rDNA. Sequences obtained for the three isolates were identical, indicating that they were the same strain. Conclusions. The first direct connection between human disease and A. griffini is reported from a case of Acanthamoeba keratitis. The type strain of this species was isolated from a marine environment, but the disease<ausing strain was isolated from a domestic water supply. The DNA sequences obtained confirmed unequivocally the epidemiologic association between a keratitis-causing strain of Acanthamoeba, the contact lens storage case, and the domestic water supply

Detection of Bacterial Endosymbionts in Clinical Acanthamoeba Isolates

PURPOSE: To determine the presence of four clinically relevant bacterial endosymbionts in Acanthamoeba isolates obtained from patients with Acanthamoeba keratitis (AK) and the possible contribution of endosymbionts to the pathogenesis of AK. DESIGN: Experimental study PARTICIPANTS: Acanthamoeba isolates (N=37) recovered from cornea and contact lens paraphernalia of 23 patients with culture proven AK and 1 environmental isolate. METHODS: Acanthamoeba isolates were evaluated for the presence of microbial endosymbionts belonging to the bacterial genera Legionella, Pseudomonas, Mycobacteria and Chlamydia using molecular techniques (Polymerase chain reaction and sequence analysis, fluorescent in situ hybridization) and transmission electron microscopy. Corneal toxicity and virulence of Acanthamoeba isolates with and without endosymbionts were compared using a cytopathic effect (CPE) assay of human corneal epithelial cells in vitro. Initial visual acuity (VA), location and characteristics of the infiltrate, time to detection of the infection and symptoms duration at presentation were evaluated in all patients. MAIN OUTCOME MEASURES: Prevalence and potential pathobiology of bacterial endosymbionts detected in Acanthamoeba isolates recovered from AK. RESULTS: Twenty-two of the 38 (59.4%) cultures examined contained at least one bacterial endosymbiont. One isolate contained two endosymbionts, Legionella and Chlamydia, confirmed by fluorescence in situ hybridization. Corneal toxicity (CPE) was significantly higher for Acanthamoebae hosting endosymbionts compared to isolates without endosymbionts (p<0.05). Corneal pathogenic endosymbionts such as Pseudomonas and Mycobacterium enhanced Acanthamoeba CPE significantly more than Legionella (p<0.05). In the presence of bacterial endosymbionts, there was a trend toward worse initial VA (p>0.05), central location (p<0.05), absence of radial perineuritis (p<0.05), delayed time to detection (p>0.05) and longer symptoms duration at presentation (p>0.05). CONCLUSION: The majority of Acanthamoeba isolates responsible for AK harbors one or more bacterial endosymbionts. The presence of endosymbionts enhances the corneal pathogenicity of Acanthamoeba isolates and might impact detection time and clinical features of AK

Cysticidal Activity of Antifungals against Different Genotypes of Acanthamoeba

Antifungal drugs have been proposed as a novel treatment for Acanthamoeba keratitis. The cysticidal activity of several antifungal compounds was tested against different genotypes of culture collection and clinical isolates of Acanthamoeba. Only voriconazole and posaconazole were found to be cysticidal, with no differences in activity observed between clinical and culture collection isolates

Drug-resistant severe Acanthamoeba keratitis caused by rare T5 Acanthamoeba genotype

To describe a case of severe and drug-resistant Acanthamoeba keratitis in a contact lens wearer caused by atypical T5 Acanthamoeba genotype (Acanthamoeba lenticulata). Report of a case, Acanthamoeba DNA amplification and sequencing. A 61-year-old patient was referred to our clinic with a 2-week history of keratitis. Acanthamoeba keratitis (AK) was diagnosed using confocal microscopy and corneal scraping culture. Using polymerase chain reaction (PCR) and DNA sequencing, the organism was classified as a T5 genotype (A. lenticulata). The keratitis continued to progress despite topical antiamoebic therapy and ultimately led to enucleation of the affected eye. T5 genotype Acanthamoeba can cause severe AK. Atypical Acanthamoeba genotypes could be associated with worse prognosis and resistance to therapy

Utility of Molecular Sequence Analysis of the ITS rRNA Region for Identification of Fusarium spp. from Ocular Sources

PURPOSE. Fungal ocular infections cause significant ocular morbidity, particularly when diagnosis and treatment are delayed. Accurate morphologic identification of Fusarium spp. beyond the genus is time-consuming and insensitive. It was the purpose of this study to examine the usefulness of the nuclear ribosomal RNA (rRNA) internal transcribed spacer regions (ITS1 and -2) to detect and differentiate Fusarium spp. responsible for ocular infections.METHODS. Fifty-eight archived isolates from ocular sources of 52 patients diagnosed with Fusarium keratitis at the Bascom Palmer Eye Institute (Miami, FL) from April 2000 to May 2007 were analyzed. the archived samples, which were initially classified according to morphologic characteristics, were analyzed by DNA sequence data generated from the ITS regions of the rRNA genes.RESULTS. Fifteen distinct sequences were identified among the 58 isolates. Sequence analysis identified the isolates as Fusarium solani (75%), F. oxysporum (16%), F. incarnatum-equiseti (5%), F. dimerum (2%), and one Fusarium sp. (2%) that was not classified within any species complex. Species identification based on sequence data correlated well with the morphologic classification when performed by a mycology reference laboratory, but a higher rate of mismatch was observed based on identification by a nonreference laboratory.CONCLUSIONS. Most of the isolates of Fusarium ocular infections belong to the F. solani or F. oxysporum species complexes. Morphologic classification at the species level yielded inconsistent results at a general microbiology laboratory. in contrast, the sequence variation within the ITS region allowed reliable and faster discrimination of the isolates at both the genus and species level. (Invest Ophthalmol Vis Sci. 2009;50:2230-2236) DOI:10.1167/iovs.08-2757National Eye InstituteBausch Lomb, Inc.Univ Miami, Miller Sch Med, Bascom Palmer Eye Inst, Miami, FL 33136 USAUniv Miami, Rosenstiel Sch Marine & Atmospher Sci, Miami, FL 33136 USAUniversidade Federal de São Paulo, Dept Ophthalmol, São Paulo, BrazilUniv So Calif, Keck Sch Med, Inst Med Genet, Los Angeles, CA 90033 USAUniversidade Federal de São Paulo, Dept Ophthalmol, São Paulo, BrazilNational Eye Institute: P30 EY014801Web of Scienc