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

Lead Research Organisation: Queen's University of Belfast
Department Name: Centre for Experimental Medicine


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.


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Description Application of microbial bioinformatics to investigate corneal infections
Amount £10,228 (GBP)
Funding ID NE/T014148/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 04/2020 
End 03/2021