Koyanagi Yoshito, Sajiki Ai Fujita, Yuki Kenya, Ushida Hiroaki, Kawano Kenichi, Fujita Kosuke, Shimizu Hideyuki, Okuda Daishi, Kosaka Mitsuki, Yamada Kazuhisa, Suzumura Ayana, Kachi Shu, Kaneko Hiroki, Komatsu Hiroyuki, Usui Yoshihiko, Goto Hiroshi, Nishiguchi Koji M
Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Department of Ophthalmology, Yokkaichi Municipal Hospital, Yokkaichi, Japan.
Invest Ophthalmol Vis Sci. 2025 Apr 1;66(4):50. doi: 10.1167/iovs.66.4.50.
To investigate the sensitivity and specificity of herpes virus detection by nanopore metagenomic analysis (NMA) compared with multiplex polymerase chain reaction (mPCR)-positive and -negative controls.
This study included 43 patients with uveitis who had been screened for intraocular herpes virus infection using mPCR from aqueous humor samples. Aqueous humor samples stored after mPCR were subjected to whole-genome amplification, long-read sequencing, and analysis of the phylogenetic microorganism composition using a Flongle flow cell on the Oxford Nanopore MinION platform. For samples that tested positive with mPCR and negative with the Flongle flow cell, additional long-read sequencing was performed using a MinION flow cell, which enabled acquisition of more sequence data. The sensitivity and specificity of herpes virus detection by NMA were compared with the mPCR-positive and -negative controls.
NMA using a Flongle flow cell detected the pathogenic virus in 60.0% of those who tested positive by mPCR (12/20). Further analysis using the MinION flow cell successfully identified viral DNA fragments in three out of the eight initially undetected samples, yielding a collective sensitivity of 75.0% (15/20). All of the virus detected with the long-read sequencing were identical to those diagnosed by mPCR testing, and none of the samples that tested negative by mPCR revealed herpes viral DNA with the use of long-read sequencing.
For the detection of etiologic herpes virus DNA fragments, NMA revealed a reasonable sensitivity and high specificity. Our study highlights the potential of nanopore sequencing to facilitate further advances in uveitis diagnosis.
与多重聚合酶链反应（mPCR）阳性和阴性对照相比，研究通过纳米孔宏基因组分析（NMA）检测疱疹病毒的敏感性和特异性。
本研究纳入了43例葡萄膜炎患者，这些患者的房水样本已使用mPCR进行了眼内疱疹病毒感染筛查。mPCR后保存的房水样本进行全基因组扩增、长读长测序，并使用牛津纳米孔MinION平台上的Flongle流动槽分析系统发育微生物组成。对于mPCR检测呈阳性但Flongle流动槽检测呈阴性的样本，使用MinION流动槽进行额外的长读长测序，从而能够获取更多序列数据。将NMA检测疱疹病毒的敏感性和特异性与mPCR阳性和阴性对照进行比较。
使用Flongle流动槽的NMA在mPCR检测呈阳性的患者中检测到致病病毒的比例为60.0%（12/20）。使用MinION流动槽进行进一步分析，在最初未检测到的8个样本中的3个样本中成功鉴定出病毒DNA片段，总体敏感性为75.0%（15/20）。通过长读长测序检测到的所有病毒均与mPCR检测诊断出的病毒相同，并且mPCR检测呈阴性的样本中没有一个通过长读长测序显示出疱疹病毒DNA。
对于病因性疱疹病毒DNA片段的检测，NMA显示出合理的敏感性和高特异性。我们的研究突出了纳米孔测序在促进葡萄膜炎诊断进一步发展方面的潜力。
