| Autor: |
Thompson, Paul P., Kowalski, Regis P., Shanks, Robert M. Q., Gordon, Y. Jerold |
| Zdroj: |
Journal of Clinical Microbiology; October 2008, Vol. 46 Issue: 10 p3232-3236, 5p |
| Abstrakt: |
ABSTRACTConfirmation of Acanthamoebakeratitis by laboratory diagnosis is the first step in the treatment of this vision-threatening disease. Two real-time PCR TaqMan protocols (the Rivie`re and Qvarnstrom assays) were developed for the detection of genus-specific AcanthamoebaDNA 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 Rivie`re and Qvarnstrom assays were 11.3 DNA copies/10 µl and 94% and 43.8 DNA copies/10 µl and 92%, respectively. Our extraction protocol enabled us to detect 0.7 Acanthamoebacysts/10 µl and 2.3 Acanthamoebatrophozoites/10 µl 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 Acanthamoebaculture negative and real-time PCR positive. Our results indicate that both real-time PCR assays could be used to diagnose Acanthamoebakeratitis. Polyhexamethylene biguanide can inhibit PCR, and we suggest that specimen collection occur prior to topical treatment to avoid possible false-negative results. |
| Databáze: |
Supplemental Index |
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