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J. Clin. Microbiol. doi:10.1128/JCM.00908-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Validation of Real-Time PCR for the Laboratory Diagnosis of Acanthamoeba Keratitis

The Charles T. Campbell Ophthalmic Microbiology Laboratory, UPMC Eye Center, Ophthalmology and Visual Sciences Research Center, University of Pittsburgh School of Medicine, Pittsburgh, PA

^* To whom correspondence should be addressed. Email: thompsonpp{at}upmc.edu.

	Abstract

Confirmation of Acanthamoeba keratitis by laboratory diagnosis is the first step in the treatment of this vision-threatening disease. Two real-time polymerase chain reaction (PCR) TaqMan protocols (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 by determining: 1) the real-time PCR limit of detection and amplification efficiency; 2) the ability to detect trophozoites and cysts, and; 3) 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/10µl, 94%, and 43.8 DNA copies/10µl, 92%, respectively. Our extraction protocol enabled us to detect 0.7 Acanthamoeba cysts/10µl and 2.3 Acanthamoeba trophozoites/10µl 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% (7/7) and 100% (37/37), 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, 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.