title: Comparison of Validated Polymerase Chain Reaction and Culture Isolation for the Routine Detection of Acanthamoeba From Ocular Samples.
abstract: OBJECTIVE
  Acanthamoeba keratitis should be definitively diagnosed for appropriate therapy. Our institution has validated polymerase chain reaction (PCR) as a routine diagnostic test to detect Acanthamoeba DNA from ocular samples. We compared PCR with culture isolation for detecting Acanthamoeba from ocular samples.
  
  
  METHODS
  The microbiology records of patients that had specimens submitted (May 2012 to January 2014) for laboratory testing for Acanthamoeba keratitis were reviewed for (1) Acanthamoeba culture isolation, (2) Acanthamoeba DNA detection by PCR, and (3) non-Acanthamoeba culture results. For Acanthamoeba isolation, corneal samples were planted on nonnutrient agar overlaid with Enterobacter aerogenes. Validated PCR (May 2012) for Acanthamoeba DNA was processed at the Division of Molecular Diagnostics, UPMC, Pittsburgh, PA. Additional cultures were obtained for bacteria, fungus, and virus (i.e., herpes simplex virus) using standard techniques.
  
  
  RESULTS
  Culture isolation and PCR were processed on 125 patients with a differential diagnosis of Acanthamoeba keratitis. Of these, 104 (83.2%) were culture negative, PCR negative; 14 (11.2%) were culture positive, PCR positive; 4 (3.2%) were culture negative, PCR positive; and, 3 (2.4%) were culture positive, PCR negative. Culture and PCR were statistically equivalent for detecting Acanthamoeba from ocular samples (P=1.0, McNemar's test). Nineteen of the culture-negative, PCR-negative corneal samples (18.3%) were positive for other pathogens such as bacteria, fungus, and virus.
  
  
  CONCLUSIONS
  There is no clear advantage of PCR over culture isolation for detecting Acanthamoeba in ocular specimens. Other pathogens such as bacteria, fungus, and virus must still be considered in severe persistent keratitis. Polymerase chain reaction seems to be a complementary test for the clinical support of Acanthamoeba keratitis.
authors.affiliation: Department of Ophthalmology (R.P.K., L.M.K., A.M.), Charles T. Campbell Eye Microbiology Laboratory, University of Pittsburgh Medical Center (UPMC), University of Pittsburgh, Pittsburgh, PA; and Division of Molecular Diagnostics (M.A.M.), Department of Pathology, University of Pittsburgh Medical Center (UPMC), University of Pittsburgh, Pittsburgh, PA.
authors.forename: Regis P
authors.initials: RP
authors.lastname: Kowalski
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Melan
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Karenchak
authors.forename: Alex
authors.initials: A
authors.lastname: Mammen
mesh_terms: D000048:Acanthamoeba / Q000302:isolation & purification*; D015823:Acanthamoeba Keratitis / Q000175:diagnosis*; D019411:Clinical Laboratory Techniques / Q000592:standards*; D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D016133:Polymerase Chain Reaction / Q000592:standards*; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial