Satoko Nakano1, Yasuhiro Tomaru2, Toshiaki Kubota1, Hiroshi Takase3, Manabu Mochizuki4, Norio Shimizu2, Sunao Sugita5. 1. Department of Ophthalmology, Oita University, Yufu, Japan. 2. Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan. 3. Department of Ophthalmology & Visual Science, Tokyo Medical and Dental University Graduate School of MedicineMedical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan. 4. Department of Ophthalmology & Visual Science, Tokyo Medical and Dental University Graduate School of MedicineMedical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan; Miyata Eye Hospital, Miyakonojo, Japan. 5. Department of Ophthalmology, Kobe City Eye Hospital, Kobe, Japan; Laboratory for Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan. Electronic address: sunaoph@cdb.riken.jp.
Abstract
PURPOSE: A novel multiplex polymerase chain reaction (PCR) test (Strip PCR) for 24 common ocular infectious disease pathogens was established. Solid-phase techniques provide stable, prompt, and accurate results while using less sample amount with lower cost than conventional quantitative real-time PCR (qPCR). Strip PCR for infectious uveitis was optimized and evaluated using intraocular samples. DESIGN: Evaluation of diagnostic testing. METHODS: We examined 722 samples at 14 institutions. Genomic DNA from aqueous humor and vitreous fluid was analyzed by qPCR and Strip PCR. Clinical diagnosis was determined based on symptoms, clinical findings, and laboratory tests. MainOutcomeMeasures: The diagnostic parameters of the Strip PCR were based on qPCR results. RESULTS: Strip PCR showed low intra- and inter-institutional variability even when performed by technicians with various PCR skill levels. The targets of Strip PCR for infectious uveitis were optimized for 9 major pathogens (herpes simplex virus [HSV] 1, HSV2, varicella-zoster virus, human T-cell lymphotropic virus 1, human herpesvirus 6, Epstein-Barr virus, cytomegalovirus, Toxoplasma gondii, and Treponema pallidum) with 772 intraocular samples. The Strip PCR successfully detected pathogen DNA at concentrations ranging from 100 to 109 copies/mL in 252 of the 255 qPCR-positive samples. It yielded negative results for all the 191 qPCR-negative samples. Strip PCR had higher sensitivity (98.8%), specificity (98.5%), positive predictive value (98.8%), and negative predictive value (98.5%) than qPCR, with distinct primers. The Strip PCR results had strong correlation with that of the qPCR (r = 0.838) and they were consistent with the clinical diagnosis. CONCLUSIONS: Easy-to-use Strip PCR is recommended for rapid diagnosis of infectious uveitis, as its results are equivalent to that of conventional qPCR.
PURPOSE: A novel multiplex polymerase chain reaction (PCR) test (Strip PCR) for 24 common ocular infectious disease pathogens was established. Solid-phase techniques provide stable, prompt, and accurate results while using less sample amount with lower cost than conventional quantitative real-time PCR (qPCR). Strip PCR for infectious uveitis was optimized and evaluated using intraocular samples. DESIGN: Evaluation of diagnostic testing. METHODS: We examined 722 samples at 14 institutions. Genomic DNA from aqueous humor and vitreous fluid was analyzed by qPCR and Strip PCR. Clinical diagnosis was determined based on symptoms, clinical findings, and laboratory tests. MainOutcomeMeasures: The diagnostic parameters of the Strip PCR were based on qPCR results. RESULTS: Strip PCR showed low intra- and inter-institutional variability even when performed by technicians with various PCR skill levels. The targets of Strip PCR for infectious uveitis were optimized for 9 major pathogens (herpes simplex virus [HSV] 1, HSV2, varicella-zoster virus, human T-cell lymphotropic virus 1, human herpesvirus 6, Epstein-Barr virus, cytomegalovirus, Toxoplasma gondii, and Treponema pallidum) with 772 intraocular samples. The Strip PCR successfully detected pathogen DNA at concentrations ranging from 100 to 109 copies/mL in 252 of the 255 qPCR-positive samples. It yielded negative results for all the 191 qPCR-negative samples. Strip PCR had higher sensitivity (98.8%), specificity (98.5%), positive predictive value (98.8%), and negative predictive value (98.5%) than qPCR, with distinct primers. The Strip PCR results had strong correlation with that of the qPCR (r = 0.838) and they were consistent with the clinical diagnosis. CONCLUSIONS: Easy-to-use Strip PCR is recommended for rapid diagnosis of infectious uveitis, as its results are equivalent to that of conventional qPCR.