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A one-step multiplex PCR for acanthamoeba keratitis diagnosis and quality samples control.

Abstract：

PURPOSE: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.

journal_name

Invest Ophthalmol Vis Sci

journal_title

Investigative ophthalmology & visual science

authors

Maubon D,Dubosson M,Chiquet C,Yera H,Brenier-Pinchart MP,Cornet M,Savy O,Renard E,Pelloux H

doi

10.1167/iovs.11-8587

subject

Has Abstract

pub_date

2012-05-14 00:00:00

pages

2866-72

issue

eissn

0146-0404

issn

1552-5783

pii

iovs.11-8587

journal_volume

pub_type

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A one-step multiplex PCR for acanthamoeba keratitis diagnosis and quality samples control.