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

Purpose: The aim of this study was to evaluate a DNA dot hybridization assay (DHA) for assessing bacterial bioburden in orthokeratology lens (OK) storage cases., Methods: Forty-one OK wearers participated in this study. The dot hybridization assay was used to assess the bacterial bioburden of OK after removal and 6-hour soaking in a storage case. Signals of the DHA were standardized after gray image transformation. The correlations between the hybridization intensities of three universal bacteria probes (BP1, BP2, and BP3) and bacterial bioburden determined by culture (colony forming units per milliliter) was analyzed by Pearson's correlation coefficient and receiver operating characteristic plots. In addition, three genus-specific probes for Pseudomonas, Acinetobacter, and Klebsiella were used to detect potentially hazardous bacterial contamination regardless of bacterial viability status., Results: Among the three universal probes, there were good correlations between probe BP2 (r2 = 0.31, P = 9.5 × 10(-5)) and probe BP3 (r2 = 0.35, P = 3.1 × 10(-5)) with bacterial bioburden, but no correlation was found between probe BP1 and bacterial bioburden (r2 = 0.04, P = 0.11). In 41 samples, one was Pseudomonas-positive by both DHA and culture, while 10 were Pseudomonas-positive by DHA but negative by culture. Furthermore, nine samples tested positive for Acinetobacter (n = 7) and Klebsiella (n = 2) by DHA only., Conclusions: The dot hybridization assay provides a novel way to assess the bacterial bioburden of OK storage cases. Lens care quality can be assessed with universal bacteria probes, while potentially hazardous bacterial contamination can be traced with genus-specific probes., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)