title: A one-step multiplex PCR for acanthamoeba keratitis diagnosis and quality samples control.
abstract: OBJECTIVE
  As the number of cases of Acanthamoeba spp. keratitis (AK) is constantly growing, new diagnostic tools are needed to confirm and guide ophthalmologists in this clinically problematic diagnosis. Molecular diagnosis is particularly well adapted, although only a few real-time PCR techniques have been described recently. The aim of this study was to develop a new PCR technique for the diagnosis of AK by combining the detection of Acanthamoeba DNA with human DNA, thus allowing an accurate interpretation of the PCR result.
  
  
  METHODS
  Different DNA extraction procedures were compared to ensure an optimized amplification of one Acanthamoeba genome. The analytical parameters of this new multiplex Acanthamoeba beta-globin PCR (MAB-PCR) were evaluated. Fourteen eye drops were tested as potential PCR inhibitors. A prospective series of 28 corneal scrapings was subjected to MAB-PCR.
  
  
  RESULTS
  The best extraction procedure associated thermal-shock pretreatment followed by a manual extraction procedure. The MAB-PCR parameters displayed excellent specificity and sensitivity, with a detection of 0.02 genome of Acanthamoeba. No eye drops were total PCR inhibitors. Of 28 corneal scrapings, 18 were considered true negatives. Seven could not be interpreted because of insufficient scraping material. Three were considered true positives: every patient progressed favorably on specific and reliable treatment.
  
  
  CONCLUSIONS
  The MAB-PCR is a new tool to diagnose AK. It allows rapid diagnosis and prompt treatment of this probably underestimated etiology of infectious keratitis. This optimized real-time PCR outperforms the gold standard for Acanthamoeba keratitis diagnosis and it allows a concomitant evaluation of the quality of the corneal scraping, which is necessary for a precise interpretation of the results.
authors.affiliation: Parasitology-Mycology Laboratory, Infectious Agents Department, Grenoble Teaching Hospital, Grenoble, France. DMaubon@chu-grenoble.fr
authors.forename: Danièle
authors.initials: D
authors.lastname: Maubon
authors.forename: Muriel
authors.initials: M
authors.lastname: Dubosson
authors.forename: Christophe
authors.initials: C
authors.lastname: Chiquet
authors.forename: Hélène
authors.initials: H
authors.lastname: Yera
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Brenier-Pinchart
authors.forename: Muriel
authors.initials: M
authors.lastname: Cornet
authors.forename: Olivier
authors.initials: O
authors.lastname: Savy
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Renard
authors.forename: Hervé
authors.initials: H
authors.lastname: Pelloux
mesh_terms: D000048:Acanthamoeba / Q000235:genetics / Q000302:isolation & purification*; D015823:Acanthamoeba Keratitis / Q000175:diagnosis*; D002478:Cells, Cultured; D003315:Cornea / Q000382:microbiology; D016054:DNA, Protozoan / Q000032:analysis; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan