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...