A Multiplex Dot Hybridization Assay For Detection and Differentiation of Acanthamoeba and Herpes Keratitis.
OBJECTIVE
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.
Department of Ophthalmology Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
Ming-Tse
MT
Kuo
Department of Ophthalmology Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
Po-Chiung
PC
Fang
Department of Ophthalmology Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
Hun-Ju
HJ
Yu
Department of Laboratory Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
Tsae-Ling
TL
Chao
Department of Laboratory Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
Chun-Chih
CC
Chien
Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Shun-Hua
SH
Chen
Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Jen-Ren
JR
Wang
Department of Ophthalmology Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
Shin-Ling
SL
Tseng
Department of Ophthalmology Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
Yu-Hsuan
YH
Lai
Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, and Center for Shockwave Medicine and Tissue Engineering, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan.
Chang-Chun
CC
Hsiao
Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Tsung C
TC
Chang
D000048:Acanthamoeba; D015823:Acanthamoeba Keratitis / Q000175:diagnosis*; D003315:Cornea / Q000469:parasitology / Q000821:virology; D003937:Diagnosis, Differential; D006801:Humans; D016849:Keratitis, Herpetic / Q000175:diagnosis*; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction; D009693:Nucleic Acid Hybridization / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D018139:Simplexvirus