Francis I. Proctor Foundation, University of California San Francisco, San Francisco, CA, USA; Department of Ophthalmology, University of California, San Francisco, CA, USA.
Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
J Clin Virol. 2021 Mar;136:104759. doi: 10.1016/j.jcv.2021.104759. Epub 2021 Feb 11.
Molecular diagnostics such as pathogen-directed PCRs have transformed testing for ocular infections since the late 1990s. Although these assays remain important diagnostic tools for samples with low biomass, the lack of diagnostic range motivates alternative molecular approaches for ocular infections. The aim of this study was to determine the performance of a high-throughput RNA sequencing approach, RNA-seq, to detect infectious agents in ocular samples from patients with presumed ocular infections.
We compared the performance of RNA-seq to pathogen-directed PCRs using remnant nucleic acids from 41 aqueous or vitreous samples of patients with presumed ocular infections. Pathogen-directed PCRs were performed at the CLIA-certified Stanford Clinical Virology Laboratory. RNA-seq was performed in a masked manner at the Proctor Foundation at the University of California San Francisco. Percent positive and negative agreement between the two testing approaches were calculated. Discordant results were subjected to orthogonal testing.
The positive percent agreement between RNA-seq and pathogen-directed PCRs was 100% (95% confidence interval (CI): 78.5%-100%). The negative percent agreement was 92.6% (95% CI: 76.6%-97.9%). RNA-seq identified pathogens not on the differential diagnosis for 9.7% (4/41) of the samples. Two pathogens solely identified with RNA-seq were confirmed with orthogonal testing.
RNA-seq can accurately identify common and rare pathogens in aqueous and vitreous samples of patients with presumed ocular infections. Such an unbiased approach to testing has the potential to improve diagnostics although practical clinical utility warrants additional studies.
自 20 世纪 90 年代末以来，分子诊断技术（如针对病原体的 PCR）改变了眼部感染的检测方式。尽管这些检测方法仍然是低生物量样本的重要诊断工具，但缺乏诊断范围促使人们寻求替代的眼部感染分子方法。本研究旨在确定高通量 RNA 测序（RNA-seq）方法在检测疑似眼部感染患者眼部分泌物中传染性病原体的性能。
我们比较了 RNA-seq 与针对病原体的 PCR 检测方法在 41 例疑似眼部感染患者的房水或玻璃体液残留核酸样本中的性能。针对病原体的 PCR 是在 CLIA 认证的斯坦福临床病毒学实验室进行的。RNA-seq 是在加利福尼亚大学旧金山分校 Proctor 基金会以盲法进行的。计算两种检测方法之间的阳性和阴性符合率。对不一致的结果进行正交检测。
RNA-seq 与针对病原体的 PCR 之间的阳性符合率为 100%（95%置信区间：78.5%-100%）。阴性符合率为 92.6%（95%置信区间：76.6%-97.9%）。RNA-seq 确定了 9.7%（4/41）的样本不在鉴别诊断中的病原体。通过正交检测证实了仅通过 RNA-seq 鉴定出的两种病原体。
RNA-seq 可以准确识别疑似眼部感染患者的房水和玻璃体液样本中的常见和罕见病原体。这种无偏倚的检测方法有可能改善诊断，但实际的临床应用价值需要进一步研究。
