A Multiplex Dot Hybridization Assay For Detection and Differentiation of Acanthamoeba and Herpes Keratitis

We verified a multiplex dot hybridization (MDH) assay for the rapid detection and differentiation of Acanthamoeba keratitis (AK) and herpes simplex keratitis (HSK).

Molecular detection of Acanthamoeba and herpes simplex virus in corneal scrapes was performed with the MDH assay and standard diagnostic methods. The experimental group included corneal scrapes (n = 33) from patients with culture- or pathology-confirmed AK (n = 15) and real-time PCR-confirmed HSK (n = 16). The control group included 50 samples from cases of bacterial keratitis (n = 15), fungal keratitis (n = 15), and initially presumed AK (n = 5) or HSK (n = 17) which finally were excluded by culture for Acanthamoeba or by real-time PCR for herpes simplex virus, respectively. Discrepant results between methods were resolved by DNA sequencing of the PCR amplicons.

After discrepant analysis, the sensitivity for the diagnosis of AK and HSK was both 93.3% by the MDH assay, while the specificity was 100% for the two types of keratitis. The turnaround time of MDH assay was within a working day using an already prepared array. Two false-negatives (one AK case and one HSK case) were obtained by the MDH assay.

The MDH assay could effectively prevent missed or delayed diagnosis of AK and HSK and has a potential to be adopted in routine clinical practice if the test is commercialized.