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Satoko Nakano, Sunao Sugita, Yasuhiro Tomaru, Takako Nakamuro, Hiroshi Takase, Norio Shimizu, Manabu Mochizuki, Toshiaki Kubota; Establishment of a new comprehensive polymerase chain reaction (PCR) strip kit to diagnose infectious eye diseases. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
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© ARVO (1962-2015); The Authors (2016-present)
To improve previously reported comprehensive PCR system (Ophthalmol., 2013;120:1761-8) and establish a new PCR kit, and to report a pilot clinical study to test its usefulness in the diagnosis of infectious eye diseases.
A multiplex solid-phase PCR strip kit was established. In each well, specific probes and primers of genomic DNA of 2-3 microorganisms were fixed, dried and stored at room temperature until use. A total of 24 common pathogens of infectious eye diseases including all 8 types of human herpes virus (HHV), HTLV1, Adenovirus, Mycobacterium Tuberculosis, Treponema pallidum, P. acnes, bacterial 16S ribosomal DNA (rDNA), Candida spp, C. glabrata, C. krusei, Aspergillus, Fusarium, fungal 28S rDNA, Toxoplasma gondi, Toxocara canis, Chlamydia and Acanthamoeba were selected for the PCR strip kit. After IRB approval was obtained, a pilot clinical study to test the usefulness of the PCR strip kit was performed using ocular samples from 22 patients with infectious eye diseases and 8 controls with non-infectious eye diseases at Oita University Hospital.
The PCR strip kit detected all genomic DNA of tested positive control microorganisms, but not negative control. In the clinical study, among 22 patients with various infectious eye diseases, HSV1 (n = 1), VZV (n = 3), CMV (n = 2), EBV (n = 3), HHV6 (n = 2), HTLV-1 (n = 3), Treponema pallidum (n = 1), bacterial 16S (n = 5), Candida spp (n = 4), Aspergillus (n = 2), Fungal 28S (n = 5) were detected, but none in control patients. The clinical presentations of PCR positive patients were in accord with those of respective infectious diseases and the results were further conformed by our previous comprehensive PCR. On the other hand, P. acnes was detected in 15/22 (68.2%) of patients and 8/8 (100.0%) in controls. Bacterial 16S was detected in 5/22 (22.7%) of patients and 6/8 (75.0%) of controls. It is of note that P ances and bacterial 16S were detected in the end of PCR cycles and found to be low concentration by quantitative real-time PCR. No pathogens other than P.acnes and bacterial 16S were detected in the control samples.
The new multiplex PCR strip can be used for rapid comprehensive diagnosis of the infectious eye diseases. A prospective multicenter clinical study is necessary to determine its usefulness in infectious eye diseases.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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