杂交捕获法二代测序技术在眼部感染病原体检测中的应用初探  

Preliminary Exploration of Hybridization Capture-Based Next-Generation Sequencing for the Detection of Ocular Infection Pathogens

作　　者：叶定兴 闫恒 刘军权 沈洋洋 徐一 王海鸥 魏文龙 郑美琴 Dingxing Ye;Heng Yan;Junquan Liu;Yangyang Shen;Yi Xu;Haiou Wang;Wenlong Wei;Meiqin Zheng(National Clinical Research Center for Ocular Diseases,Eye Hospital,Wenzhou Medical University,Wenzhou 325027,China;Hangzhou Kingmed Medical Laboratory Co.,Ltd,Hangzhou 310000,China)

机构地区：[1]温州医科大学附属眼视光医院国家眼耳鼻喉疾病(眼部疾病)临床医学研究中心,温州325027 [2]杭州金域医学检验实验室有限公司,杭州310000

出　　处：《中华眼视光学与视觉科学杂志》2025年第1期36-45,共10页Chinese Journal Of Optometry Ophthalmology And Visual Science

基　　金：温州市科技局重大科研创新攻关项目(ZX-ZY2022018);温州市科技局基础性科研项目(Y2023789)。

摘　　要：目的:初步探讨基于杂交捕获法的二代测序技术在眼部感染病原体检测中的应用价值。方法:回顾性系列病例研究。随机纳入2021年7月至2023年11月温州医科大学附属眼视光医院确诊为眼部感染且传统病原学检测结果为阳性的入院患者17例(17眼)。所有患者当时收治时根据感染部位采集结膜囊分泌物和角膜刮取物或玻璃体液样本,进行涂片镜检与培养鉴定,剩余样本进行了宏基因组测序(mNGS)和杂交捕获法二代测序(HC-NGS),对3种方法检测到的病原体种类和数量进行分析,并对病原体检出率、混合感染率、病原体种类一致性等进行分析。比较了mNGS和HC-NGS在归一化序列数与耐药基因上的差异。采用费希尔精确检验比较3种方法的检出率和混合感染率差异,采用秩和检验比较分析mNGS和HC-NGS归一化序列数差异。结果:涂片镜检与培养鉴定、mNGS和HC-NGS分别鉴定出病原体18、31、39株;细菌检出率分别为35%、41%、47%;真菌检出率分别为59%、41%、41%;病毒检出率分别为6%、53%和65%。与涂片镜检和培养鉴定相比,HC-NGS和mNGS明显提高了病毒检出率(χ^(2)=7.70,P=0.008;χ^(2)=12.88,P=0.001)。涂片镜检与培养鉴定、mNGS、HC-NGS检出的混合感染率分别为6%、47%和53%,后2种方法的检出率明显高于前者(χ^(2)=7.40,P=0.017;χ^(2)=9.07,P=0.007)。涂片镜检与培养鉴定、mNGS、HC-NGS检测到的病原体种类分别为14、23和29种。病原体种类一致性方面,18%的样本在3种方法中完全一致;35%的样本在3种方法中部分一致,且HC-NGS和mNGS检测出的病原体包含了通过涂片镜检与培养鉴定检测到的所有病原体;35%的样本在HC-NGS和mNGS之间完全一致;12%的样本在HC-NGS和mNGS之间具有部分一致,且前者包含后者检测到的所有病原体。此外,HC-NGS比mNGS具有更多的归一化序列数(W=496.00,P<0.001),并且检测到更多的耐药基因。结论:HC-NGS在眼部感染病原体检Objective:To initially investigate the application value of hybridization capture-based next-generation sequencing in the detection of ocular infection pathogens.Methods:This was a retrospective series case study.From July 2021 to November 2023,totally 17 patients(17 eyes),randomly selected,who were admitted to the Eye Hospital,Wenzhou Medical University and diagnosed with ocular infections with positive results from traditional pathogenic detection were included in this study.Samples of conjunctival sac secretions,corneal scrapings,or vitreous humor were collected from all patients at admission,based on the site of infection.These samples were subjected to smear microscopy,culture,and identification.The remaining samples underwent metagenomics next-generation sequencing(mNGS),and hybridization capture-based next-generation sequencing(HC-NGS).A comprehensive analysis was conducted on the types and quantities of pathogens detected by these three methodologies,focusing on factors such as pathogen detection rate,mixed infection rate,and consistency in pathogen types.Additionally,a comparison was made between mNGS and HC-NGS regarding differences in normalized sequence counts and drug resistance genes.The detection rates and mixed infection rates of the three methods were compared using Fisher's exact test,while the normalized sequence counts of mNGS and HC-NGS were compared using the rank sum test.Results:This study identified 18,31 and 39 pathogens using smear microscopy and culture,HC-NGS and mNGS respectively.The bacterial detection rates were 35%,41%,and 47%,respectively.The fungal detection rates were 59%,41%,and 41%,respectively.And the virus detection rates were 6%,53%,and 65%,respectively.Compared with smear microscopy and culture,HC-NGS and mNGS significantly improved the virus detection rate(χ^(2)=7.70,P=0.008;χ^(2)=12.88,P=0.001).The detection rates of mixed infections using smear microscopy and culture,mNGS,and HC-NGS were 6%,47%,and 53%,respectively.The detection rates of the latter two methods were s

关 键 词：病原体 眼部感染 杂交捕获法 二代测序 

分 类 号：R77[医药卫生—眼科] R446.5[医药卫生—临床医学]