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Tatsuhiko Kobayashi, Takashi Suzuki, YUKINOBU OKAJIMA, Yuichi Hori; Metagenome techniques for detection of pathogens causing ocular infection. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3686. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
It is important to detect pathogens for diagnosis of ocular infection. However pathogens cannot be sometimes detected due to less sensitivity of conventional methods including culture method. In recent years, gene amplification techniques such as PCR is effective to detect pathogen DNA with high sensitivity, but is difficult to detect unexpected pathogen. If pathogens causing ocular infection include “unknown” or “non-culturable” organism, it is difficult to understand etiology. Metagenomic analysis using clinical specimens can detect comprehensively DNA of organism including not only bacteria and fungi but also DNA virus in one time. In this study we performed metagenomic analysis of ocular specimens, and compared other examination.
The study involved 11 keratitis cases, 4 iridocyclitis cases, and one endophthalmitis case, and corneal scraping, aqueous humor, or vitreous humor, respectively, were collected. Ocular specimens was used for bacterial and fungal culture, and/or PCR detecting viral DNA including herpes simplex virus-1 (HSV-1), varicella zoster virus, and cytomegalovirus. After DNA extraction of specimens, shotgun metagenomic sequencing for 150 base pair was performed by Illumina MiSeq® System. Human genome was removed by in silico, and sequence were retrieved from database at NCBI using a MegaBLAST search.
Seven cases (43.8%) were positive for culture or PCR. Metagenome techniques revealed that 13 cases (81.3%) could include microbial genome in specimens. Five cases (31.3%) possessed genome of organism same to them which was detected by culture and PCR (. Three cases (18.8%) were negative for culture, PCR, and metagenome analysis. Whereas 7 cases (43.8%) were positive for only metagenome analysis, and they included genome of organism which were rare as causative agents of ocular infection, e.g. Prevotella sp. or Paracoccus sp.. Moreover genome of both Fusarium sp. and HSV-1 were detected in one keratitis case. However metagenome analysis showed genome of Propiobacterium acnes which might be ocular flora was found in 10 cases (62.5%, 8 keratitis and 2 iricdocyclitis cases).
Metagenome analysis using ocular sample could detect comprehensively microbial genome which was not detected by conventional examination. However attention should be paid to avoid microbial contamination when specimens are collected.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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