title: Assessment of real-time polymerase chain reaction detection of Acanthamoeba and prognosis determinants of Acanthamoeba keratitis.
abstract: OBJECTIVE
  To evaluate the diagnostic value of real-time polymerase chain reaction (PCR) for detecting Acanthamoeba in eyes diagnosed with Acanthamoeba keratitis (AK) by conventional tests. In addition, to determine the preoperative prognosis-determining factors in eyes with AK.
  
  
  METHODS
  Retrospective, cross-sectional study.
  
  
  METHODS
  A total of 104 eyes of 103 patients who were diagnosed with AK or with bacterial or bacteria-associated keratitis (BK) by conventional tests.
  
  
  METHODS
  Twenty-nine eyes with AK and 75 eyes with BK were evaluated for Acanthamoeba and bacterial DNA by real-time PCR. The Acanthamoeba copy numbers, bacterial load, and clinical parameters in the patients with AK were assessed for those significantly associated with poor outcome, that is, final visual acuity of <20/50 or requiring keratoplasty, by logistic regression analysis.
  
  
  METHODS
  Acanthamoeba DNA copy number, bacterial DNA copy number, and odds ratio (OR) for poor prognosis.
  
  
  RESULTS
  The detection of amoebic DNA was 50 times more sensitive by real-time PCR than by conventional cyst counting. The Acanthamoeba copy numbers at the first visit (mean: 4.7×10(5)±3.2×10(5) copies) were significantly correlated with the AK stage, and both were significant risk factors for a poor outcome. The Acanthamoeba DNA copy numbers at the first visit and AK stage had a significantly high risk for poor outcome (OR of Acanthamoeba DNA copy per logarithm of copy numbers: 3.48, 95% confidence interval [CI], 1.04-111.63, P<0.05; OR of AK stage: 2.8 per stage increase, 95% CI, 1.07-7.30, P<0.05, after adjustment of age). In the AK cases with poor outcome, the amoebic DNA was not reduced by more than 90% after 1 month of treatment. The weak amoebic reduction was significantly associated with advanced AK stages or previous use of steroids. Bacterial 16S rDNA was detected in 53.6% of the eyes with AK, but it was not associated with any risk for refractoriness.
  
  
  CONCLUSIONS
  Real-time PCR was effective in detecting and managing AK. The Acanthamoeba copy number and AK stage at the first visit were significantly associated with poor outcome.
  
  
  BACKGROUND
  The author(s) have no proprietary or commercial interest in any materials discussed in this article.
authors.affiliation: Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Tottori, Japan. yoshifumiikeda@hotmail.com
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Ikeda
authors.forename: Dai
authors.initials: D
authors.lastname: Miyazaki
authors.forename: Keiko
authors.initials: K
authors.lastname: Yakura
authors.forename: Asako
authors.initials: A
authors.lastname: Kawaguchi
authors.forename: Ryoko
authors.initials: R
authors.lastname: Ishikura
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Inoue
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Mito
authors.forename: Atsushi
authors.initials: A
authors.lastname: Shiraishi
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Ohashi
authors.forename: Shiro
authors.initials: S
authors.lastname: Higaki
authors.forename: Motoki
authors.initials: M
authors.lastname: Itahashi
authors.forename: Masahiko
authors.initials: M
authors.lastname: Fukuda
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Shimomura
authors.forename: Kenji
authors.initials: K
authors.lastname: Yagita
mesh_terms: D000048:Acanthamoeba / Q000235:genetics* / Q000302:isolation & purification; D015823:Acanthamoeba Keratitis / Q000175:diagnosis* / Q000469:parasitology; D000328:Adult; D003315:Cornea / Q000469:parasitology / Q000473:pathology; D003320:Corneal Ulcer / Q000175:diagnosis / Q000382:microbiology; D003430:Cross-Sectional Studies; D056915:DNA Copy Number Variations; D016054:DNA, Protozoan / Q000032:analysis*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D059208:Parasite Load; D011237:Predictive Value of Tests; D011379:Prognosis; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D014792:Visual Acuity / Q000502:physiology
chemical_list: D016054:DNA, Protozoan