title: Validation of real-time PCR for laboratory diagnosis of Acanthamoeba keratitis.
abstract: Confirmation of Acanthamoeba keratitis by laboratory diagnosis is the first step in the treatment of this vision-threatening disease. Two real-time PCR TaqMan protocols (the Rivière and Qvarnstrom assays) were developed for the detection of genus-specific Acanthamoeba DNA but lacked clinical validation. We have adapted these assays for the Cepheid SmartCycler II system (i) by determining their real-time PCR limits of detection and amplification efficiencies, (ii) by determining their ability to detect trophozoites and cysts, and (iii) by testing a battery of positive and negative samples. We also examined the inhibitory effects of a number of commonly used topical ophthalmic drugs on real-time PCR. The results of the real-time PCR limit of detection and amplification efficiency of the Rivière and Qvarnstrom assays were 11.3 DNA copies/10 microl and 94% and 43.8 DNA copies/10 microl and 92%, respectively. Our extraction protocol enabled us to detect 0.7 Acanthamoeba cysts/10 microl and 2.3 Acanthamoeba trophozoites/10 microl by both real-time PCR assays. The overall agreement between the assays was 97.0%. The clinical sensitivity and specificity of both real-time PCR assays based on culture were 100% (7 of 7) and 100% (37 of 37), respectively. Polyhexamethylene biguanide was the only topical drug that demonstrated PCR inhibition, with a minimal inhibitory dilution of 1/640 and an amplification efficiency of 72.7%. Four clinical samples were Acanthamoeba culture negative and real-time PCR positive. Our results indicate that both real-time PCR assays could be used to diagnose Acanthamoeba keratitis. Polyhexamethylene biguanide can inhibit PCR, and we suggest that specimen collection occur prior to topical treatment to avoid possible false-negative results.
authors.affiliation: The Charles T. Campbell Ophthalmic Microbiology Laboratory, Ophthalmology and Visual Sciences Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA. thompsonpp@upmc.edu
authors.forename: Paul P
authors.initials: PP
authors.lastname: Thompson
authors.forename: Regis P
authors.initials: RP
authors.lastname: Kowalski
authors.forename: Robert M Q
authors.initials: RM
authors.lastname: Shanks
authors.forename: Y Jerold
authors.initials: YJ
authors.lastname: Gordon
mesh_terms: D000048:Acanthamoeba / Q000235:genetics* / Q000302:isolation & purification; D015823:Acanthamoeba Keratitis / Q000175:diagnosis*; D000818:Animals; D001645:Biguanides / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D010274:Parasitology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D001645:Biguanides; D017931:DNA Primers; D004791:Enzyme Inhibitors; C031233:polihexanide