Development of a molecular diagnostic assay for infectious keratitis and corneal ulcer

开发感染性角膜炎和角膜溃疡的分子诊断方法

• 批准号: 1981466
• 金额: --
• 依托单位: Queen's University Belfast
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1981466
• 关键词: Development; molecular; diagnostic; assay; infectious

Corneal opacities caused by ulcers are responsible for approximately 5% of visual impairment cases worldwide, making them one of the leading causes of blindness, along with conditions such as glaucoma and cataracts. They may be caused by a very broad spectrum of pathogens and it is essential to quickly and accurately identify the specific pathogen to determine the most appropriate treatment option. However, identification of the pathogens associated with corneal ulcer (infectious keratitis) is challenging because the "corneal scrapes" traditionally collected using sterile scalpel blades are very small and only approximately half are culture-positive. It has been demonstrated that alternative samples collected using "corneal impression membranes (CIM)" can improve culture rates. The aim of this project is to combine a molecular approach with the use of CIMs to better characterise the spectrum of pathogens responsible for corneal ulcer and develop a rapid diagnostic assay.The first phase of this project will focus on microbial metagenome analysis to identify and characterise pathogens associated with serious corneal infections. Use of this molecular approach is critical because our knowledge of the aetiology of these infections based on culture is very incomplete. Both the poor isolation rate and delay in culture mean that a molecular diagnostic method is much needed. Conventional sample handling and DNA extraction techniques will be optimised as necessary to obtain microbial genomic DNA from corneal specimens. Established high-throughput sequencing methods for low input DNA (i.e. Illumina Nextera XT sequencing) will be used within the Queen's University Belfast Genomics unit to generate comprehensive metagenome data from the samples. Work to evaluate whole-genome amplification and methods to deplete specimens of non-target DNA will be undertaken, with potential for the student to develop novel methods for processing corneal specimens for molecular testing. This project will also make use of the established computational infrastructure within Queen's University in order to carry out bioinformatic analyses on the sequencing data. This will enable definition of a target gene list for development of specific molecular assays in Phase 2. Use of whole-genome sequencing (as opposed to 16S microbiome analysis) means that as a secondary objective, we can also investigate whether antibiotic sensitivity of important corneal pathogens can be predicted directly from genome data (indeed we will initially sequence pure isolates which have been cultured from patients with infectious keratitis to investigate their antibiotic susceptibility and pathogenic mechanisms). In phase 2 we will work in collaboration with our industry partner (Hibergene Diagnostics Ltd.) to develop a panel of quantitative real-time PCR reference assays based on the target list and capable of identifying the causative micro-organism. These will be optimised to serve as "Gold Standard" reference assays. A panel of rapid isothermal LAMP assays will then be developed, and the technical performance of these compared to the reference methods. If prediction of antimicrobial sensitivity directly from sequence data is feasible, then specific assays to detect key sensitivity markers will also be developed and evaluated. Finally, a clinical evaluation of the panel of LAMP assays will be undertaken using a second cohort of corneal samples. This project will encompass a broad range of biological disciplines and the student will gain skills in areas including practical molecular biology, microbiology, genomics and bioinformatics. There is a national shortage of skilled bioinformaticians and with a considerable data analysis component and the opportunity to develop custom scripts for specific analyses, this project will provide valuable training in this field.

由溃疡引起的角膜混浊是全球约5%的视力损害病例的原因，使其成为失明的主要原因之一，沿着青光眼和白内障等病症。它们可能由非常广泛的病原体引起，因此必须快速准确地识别特定病原体，以确定最合适的治疗方案。然而，与角膜溃疡（感染性角膜炎）相关的病原体的鉴定是具有挑战性的，因为传统上使用无菌手术刀刀片收集的“角膜刮擦”非常小，并且只有大约一半是培养阳性的。已经证明，使用“角膜印模膜（CIM）”收集的替代样品可以提高培养率。本项目的目的是将联合收割机与CIM结合起来，更好地鉴定角膜溃疡病原体谱，并开发快速诊断方法。本项目的第一阶段将集中于微生物宏基因组分析，以鉴定和鉴定与严重角膜感染相关的病原体。使用这种分子方法是至关重要的，因为我们的知识，这些感染的病原学的基础上文化是非常不完整的。分离率低和培养延迟都意味着急需一种分子诊断方法。常规的样本处理和DNA提取技术将根据需要进行优化，以从角膜标本中获得微生物基因组DNA。已建立的低输入DNA的高通量测序方法（即Illumina Nextera XT测序）将在女王大学贝尔法斯特基因组学单位内使用，以从样本中生成全面的宏基因组数据。将开展评估全基因组扩增和消耗非靶DNA样本的方法的工作，并有可能为学生开发处理角膜样本进行分子检测的新方法。该项目还将利用皇后大学内已建立的计算基础设施，对测序数据进行生物信息学分析。这将使得能够定义靶基因列表，用于在II期中开发特定分子测定。使用全基因组测序（与16S微生物组分析相反）意味着作为次要目标，我们还可以研究是否可以直接从基因组数据预测重要角膜病原体的抗生素敏感性（实际上，我们将首先对从感染性角膜炎患者培养的纯分离株进行测序，以研究其抗生素敏感性和致病机制）。在第二阶段，我们将与行业合作伙伴（Hibergene Diagnostics Ltd.）合作根据目标清单开发一套能够识别致病微生物的定量实时PCR参考检测方法。这些将被优化，以作为“金标准”参考检测。然后将开发一组快速等温LAMP测定，并将其技术性能与参考方法进行比较。如果直接从序列数据预测抗菌药物敏感性是可行的，则还将开发和评价检测关键敏感性标志物的特定测定。最后，将使用第二组角膜样本对LAMP检测试剂盒进行临床评价。该项目将涵盖广泛的生物学科，学生将获得实用分子生物学，微生物学，基因组学和生物信息学等领域的技能。全国缺乏熟练的生物信息学家，该项目有相当多的数据分析组成部分，并有机会为具体分析开发定制脚本，将在这一领域提供宝贵的培训。