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Pathogenic strains of the genus Acanthamoeba are causative agents of a serious sightthreatening infection of the eye known as Acanthamoeba keratitis. The prevalence of this infection has risen in the past 20 years, mainly due to the increase in number of contact lens wearers. In this study, the prevalence of Acanthamoeba in a risk group constituted by asymptomatic contact lens wearers from Tenerife, Canary Islands, Spain, was evaluated. Contact lenses and contact lens cases were analysed for the presence of Acanthamoeba isolates. The isolates' genotypes were also determined after rDNA sequencing. The pathogenic potential of the isolated strains was subsequently established using previously described molecular and biochemical assays, which allowed the selection of three strains with high pathogenic potential. Furthermore, the sensitivity of these isolates against two standard drugs, ciprofloxacin and chlorhexidine, was analysed. As the three selected strains were sensitive to chlorhexidine, its activity and IC 50 were evaluated. Chlorhexidine was found to be active against these strains and the obtained IC 50 values were compared to the concentrations of this drug present in contact lens maintenance solutions. It was observed that the measured IC 50 was higher than the concentration found in these maintenance solutions. Therefore, the ineffectiveness of chlorhexidine-containing contact lens maintenance solutions against potentially pathogenic strains of Acanthamoeba is demonstrated in this study. INTRODUCTIONFree-living amoebae of the genus Acanthamoeba are ubiquitous protozoans that pervade the entire environment and include amphizoic strains that are pathogenic to humans and animals (Schuster & Visvesvara, 2004a). These protozoans are opportunistic causal agents of a sightthreatening ulceration of the cornea called Acanthamoeba keratitis (AK), disseminated infections (mostly cutaneous and nasopharyngeal) and a usually fatal granulomatous amoebic encephalitis (Khan, 2003(Khan, , 2006Marciano-Cabral & Cabral, 2003;Schuster & Visvesvara, 2004a).AK in developed countries is often associated with inadequate care of the contact lenses and also as a consequence of corneal trauma whereas in developing nations most cases occur as a result of ocular trauma (Seal et al., 1999;Ibrahim et al., 2007;Ozkoc et al., 2008). AK symptoms are nonspecific and can be misdiagnosed as a viral, bacterial or fungal keratitis. Thus an early diagnosis is required to achieve a successful therapeutic outcome (Martínez & Visvesvara, 1991;Lorenzo-Morales et al., 2007). Currently, the recommended treatment regimen includes a biguanide [0.02 % polyhexamethylene biguanide (PHMB) or 0.02 % chlorhexidine digluconate] together with a diamidine (0.1 % propamidine isethionate, also known as Brolene, or 0.1 % hexamidine, also known as Desomedine) (Khan, 2006). Biguanides are most often used due to their excellence in the treatment of AK and are frequently combined with a diamidine due to their presumed additive anti-amoebic effect (Hay et al., 1994;Sch...

f Acanthamoeba is an opportunistic pathogen in humans, whose infections most commonly manifest as Acanthamoeba keratitis or, more rarely, granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba, they are generally lengthy and/or have limited efficacy. Therefore, there is a requirement for the identification, validation, and development of novel therapeutic targets against these pathogens. Recently, RNA interference (RNAi) has been widely used for these validation purposes and has proven to be a powerful tool for Acanthamoeba therapeutics. Ergosterol is one of the major sterols in the membrane of Acanthamoeba. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, one of the precursors for the production of cholesterol in humans and ergosterol in plants, fungi, and protozoa. Statins are compounds which inhibit this enzyme and so are promising as chemotherapeutics. In order to validate whether this enzyme could be an interesting therapeutic target in Acanthamoeba, small interfering RNAs (siRNAs) against HMG-CoA were developed and used to evaluate the effects induced by the inhibition of Acanthamoeba HMG-CoA. It was found that HMG-CoA is a potential drug target in these pathogenic free-living amoebae, and various statins were evaluated in vitro against three clinical strains of Acanthamoeba by using a colorimetric assay, showing important activities against the tested strains. We conclude that the targeting of HMG-CoA and Acanthamoeba treatment using statins is a novel powerful treatment option against Acanthamoeba species in human disease.

cMembers of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a lifethreatening encephalitis. In order to treat those infections properly, it is necessary to target the treatment not only to the trophozoite but also to the cyst. Furthermore, it may be advantageous to avoid parasite killing by necrosis, which may induce local inflammation. We must also avoid toxicity of host tissue. Many drugs which target eukaryotes are known to induce programmed cell death (PCD), but this process is poorly characterized in Acanthamoeba. Here, we study the processes of programmed cell death in Acanthamoeba, induced by several drugs, such as statins and voriconazole. We tested atorvastatin, fluvastatin, simvastatin, and voriconazole at the 50% inhibitory concentrations (IC 50 s) and IC 90 s that we have previously established. In order to evaluate this phenomenon, we investigated the DNA fragmentation, one of the main characteristics of PCD, with quantitative and qualitative techniques. Also, the changes related to phosphatidylserine exposure on the external cell membrane and cell permeability were studied. Finally, because caspases are key to PCD pathways, caspase activity was evaluated in Acanthamoeba. All the drugs assayed in this study induced PCD in Acanthamoeba. To the best of our knowledge, this is the first study where PCD induced by drugs is described quantitatively and qualitatively in Acanthamoeba.

The free-living amoeba Balamuthia mandrillaris can cause fatal encephalitis in humans and other mammals. The organism is associated with soils, and soil exposure has been identified as a risk factor for this pathogen. However, B. mandrillaris has been isolated only once from soils believed to be the source of the infection in child from California, USA who died of Balamuthia amoebic encephalitis and once from another unrelated soil source. We report for a third time the isolation of B. mandrillaris from the environment and for the second time its isolation from a sample not known to be involved with pathogenicity. We have established the new clonal B. mandrillaris strain (ID-19) in axenic media. The identity of our isolate was originally by morphology using a light microscope and this has been confirmed by 16S rRNA gene PCR. The new strain ID-19 groups with others of the species. The fact that our isolate came from dust particles deposited on surfaces from the air in an urban environment may suggest that it is not just soil exposure that constitutes a risk factor for Balamuthia infection. This is the first report of this organism from Iran.

Genotype T4 is by far the most frequent genotype of Acanthamoeba keratitis (AK) and therefore has been considered the most virulent. This study included 14 cases of AK of genotype T4 and three cases of non-T4 genotype. We found that cases of non-T4 genotype had a worse response to medical therapy, greater need for surgical intervention, greater risk of extracorneal involvement, and remarkably poorer final visual outcome than those of T4 genotype, suggesting an association between Acanthamoeba virulence and genotype that requires additional case investigation.A canthamoeba keratitis (AK) is a potentially blinding corneal infection in healthy immunocompetent individuals and is caused by free-living amoebae of the genus Acanthamoeba (1). Analysis of the 18S rRNA gene has identified 18 genotypes (T1 through T18) within this genus (2-4). Isolates from eight of the genotypic clades (T2, T3, T4, T5, T6, T10, T11, and T15) are confirmed causative agents of AK (4-12). Disease severity can range from relatively minor epithelial and subepithelial diseases, which respond well to treatment, to refractory disease that can provoke corneal melting and perforation requiring therapeutic or tectonic corneal grafts (13). Currently, the most important factors affecting the prognosis are disease severity at presentation and the interval between symptom onset and the start of effective therapy (14, 15); beginning therapy Ͼ3 weeks after symptom onset is associated with a worse prognosis.This study aimed to identify the genotypes of Acanthamoeba isolates from AK patients at our institution and to find any association between genotype and clinical outcomes.This revision was approved by the institutional review board at Hospital Universitario Ramón y Cajal. Seventeen amoebal isolates were obtained from the eyes of 17 patients with a clinical diagnosis of AK and who were followed prospectively at Hospital Universitario Ramón y Cajal, Madrid, Spain, from July 2009 to July 2013. The best spectacle-corrected visual acuity (BSCVA) in decimal Snellen fraction notation (1.0 indicates a normal value, and 0 indicates no vision at all), slit lamp and fundus examinations, and other events were recorded in the clinical notes. Tissue was obtained from each patient by epitheliectomy or by scraping the corneal ulcer when there was no epithelium left. A portion of the sample was routinely plated in blood agar, thioglycolate broth, or Sabouraud agar to rule out bacterial or fungal infection. The rest of the sample was submerged in a microcentrifuge tube with saline and sent together with the patient's contact lenses and cases to the laboratory at the University Institute of Tropical Diseases and Public Health of the Canary Islands; there it was cultured on 2% nonnutrient agar (NNA) plates and examined using an inverted microscope as previously described (16). Lenses were directly cultured in NNA and checked for amoebic growth. The contact lens cases were washed twice with 2 ml of sterile saline, and the saline was then filtered using a vacuum manifold sys...