Infectious keratitis: a molecular and clinical study

Otri, Ahmad Muneer (2013) Infectious keratitis: a molecular and clinical study. PhD thesis, University of Nottingham.

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Abstract

Infectious keratitis is a sight threatening disease which can cause permanent visual loss if not diagnosed and treated at an early stage. It can be caused by different types of microbes which are either commensals or transferred from the environment. To fight against these threats, the ocular surface (OS) has developed innate and adaptive immune mechanisms. Antimicrobial peptides (AMPs) are natural effectors on the OS with actions that range from microbicidal effects to cell signalling. Human beta defensin (hBD) 1-3 and 9, Liver expressed antimicrobial peptide (LEAP) 1 and 2, human cathelicidin (LL37), ribonuclease7 (RNase7) are the main AMPs on the OS.

In this work, the pattern of ocular AMPs gene expression in human OS cells treated with Acanthamoeba castellanii, Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) was studied and established. This was examined by quantitative real-time PCR (RT-PCR) using the Taqman assay. Among the studied AMPs, hBD3 gene showed the most significant increase in human OS cells infected with Acanthamoeba. LL37 demonstrated the highest level of gene expression in the samples infected with bacteria.

In a different study, the gene expression of two AMPs (hBD3 and 9) was studied in OS samples taken from patients with different types of infectious keratitis both during and after the infection. This was compared with the expression in healthy subjects. Impression cytology (IC) was used to obtain samples of OS epithelium from recruited subjects. An optimized method for RNA extraction of IC samples was developed. Corresponding to the results of the in vitro study, hBD3 showed an overall up-regulation in all categories whereas hBD9 was down-regulated. These changes were most significant in patients with acute Acanthamoeba keratitis. The gene expression of both hBD3 and 9 showed a tendency towards returning to the levels found in healthy subjects when healing of the corneal infection was complete.

In another study carried out to examine the antimicrobial activity of hBD3 we were surprised to find that we could not replicate this. We were unable to reproduce the previously reported antimicrobial activity of hBD3 but were able to demonstrate that the antimicrobial effect could be attributed to the acidic solvent used in preparing the hBD3 protein.

The clinical significance of application of corneal densitometry as measured by the Pentacam system was assessed for the first time in patients with infectious keratitis. We demonstrated that corneal densitometry varied with levels of inflammation and was not confined to the site of infection only. It affected the whole cornea and reverted towards normal values as the inflammation settled when the infection was brought under control. We were able to demonstrate that densitometry can be used as a measure of the corneal response to infection and inflammation and could be used to monitor response to therapy.

Finally, separate comprehensive prospective and retrospective studies of the clinical profile of severe infectious keratitis in Nottinghamshire were conducted. These two studies covered a total period of 7 years. The results of both studies were similar. Indeed, OS disease, CL wear and previous ocular surgery were found to be the most common risk factors. Positive results of corneal scraping were obtained only in about 40% of cases. Acanthamoeba, S. aureus, and P. aeruginosa were the most frequent causative organisms. Fortified topical antibiotics were effective in treating most cases. Therapeutic corneal grafting was found to be an effective and safe procedure in refractive infectious keratitis.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Dua, H.S.
Hopkinson, A.
Subjects: W Medicine and related subjects (NLM Classification) > WW Ophthalmology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
UK Campuses > Faculty of Medicine and Health Sciences > School of Clinical Sciences
Item ID: 13408
Depositing User: EP, Services
Date Deposited: 12 Feb 2014 09:14
Last Modified: 16 Sep 2016 12:54
URI: http://eprints.nottingham.ac.uk/id/eprint/13408

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