1. A DNA dot hybridization model for molecular diagnosis of parasitic keratitis.
- Author
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Huang FC, Hsieh HY, Chang TC, Su SL, Tseng SL, Lai YH, and Kuo MT
- Subjects
- Acanthamoeba genetics, Acanthamoeba Keratitis parasitology, Corneal Ulcer diagnosis, Corneal Ulcer microbiology, DNA, Fungal genetics, Eye Infections, Fungal diagnosis, Eye Infections, Fungal microbiology, Eye Infections, Parasitic parasitology, False Negative Reactions, Humans, Microsporidia genetics, Microsporidiosis diagnosis, Microsporidiosis microbiology, Molecular Diagnostic Techniques, Oligonucleotide Probes chemistry, Polymerase Chain Reaction, Predictive Value of Tests, Prospective Studies, RNA, Ribosomal genetics, RNA, Ribosomal, 18S genetics, Ribosome Subunits, Small genetics, Sensitivity and Specificity, Acanthamoeba Keratitis diagnosis, DNA, Protozoan genetics, Eye Infections, Parasitic diagnosis, Nucleic Acid Hybridization methods
- Abstract
Purpose: Developing a DNA dot hybridization model for diagnosing parasitic keratitis., Methods: Newly designed oligonucleotide probes for detecting Acanthamoeba and microsporidia were tested with target reference strains of Acanthamoeba (n = 20) and microsporidia (n = 3), and non-target microorganisms, including bacteria (n = 20) and fungi (n = 20). These probes, which had passed the preliminary tests, were then assembled as a parasite dot hybridization (PDH) model for assessing 33 clinical samples from patients with clinically suspected Acanthamoeba and microsporidia keratitis, including eight positives for Acanthamoeba , 13 positives for microsporidia, and 12 negatives for both pathogens., Results: Two probes for detecting Acanthamoeba and two for detecting microsporidia passed the tests using target and non-target strains and then were assembled in the PDH model. For clinical samples, one Acanthamoeba -positive sample (proved with pathology) was falsely negative according to the PDH assay. The sensitivity and specificity of the PDH assay for diagnosing Acanthamoeba keratitis were 87.5% and 100%, respectively, while the sensitivity and specificity for diagnosing microsporidia keratitis were 100%. The infectious agent of all clinical samples of microsporidia keratitis was identified as Vittaforma corneae with DNA sequencing, while those of Acanthamoeba keratitis were caused by four species of Acanthamoeba , with Acanthamoeba castellanii found in four samples (50%, 4/8)., Conclusions: The PDH model has the potential to be a molecular assay for diagnosing Acanthamoeba and microsporidia keratitis. However, a prospective clinical study might be needed before the model is adopted in routine clinical practice.
- Published
- 2017