Autofluorescence Signatures of Seven Pathogens: Preliminary in Vitro Investigations of a Potential Diagnostic for Acanthamoeba Keratitis.
OBJECTIVE
Acanthamoeba keratitis can cause devastating damage to the human cornea and is often difficult to diagnose by routine clinical methods. In this preliminary study, we investigated whether Acanthamoeba may be distinguished from other common corneal pathogens through its autofluorescence response. Although only a small number of pathogens were studied, the identification of a unique Acanthamoeba signature would indicate that autofluorescence spectroscopy as a diagnostic method merits further investigation.


METHODS
Samples of 7 common pathogens (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Elizabethkingia miricola, Achromobacter ruhlandii, Candida albicans, and Acanthamoeba castellanii) in solution were excited with ultraviolet light at a number of successive, narrow wavebands between 260 and 400 nm, and their fluorescence response recorded. Principal Component Analysis was used to allow better visualization of the differences in response to UV light for different species.


RESULTS
Acanthamoeba was found to possess a characteristic autofluorescence response and was easily distinguished from E. coli, S. aureus, P. aeruginosa, E. miricola, A. ruhlandii, and C. albicans over a wide range of excitation wavelengths. We also found a clear discrimination between E. coli, C. albicans, and P. aeruginosa at an excitation wavelength of 274 nm, whereas E. miricola, S. aureus, and A. ruhlandii could be separated using an excitation wavelength of 308 nm.


CONCLUSIONS
Our results, although preliminary, indicate that autofluorescence spectroscopy shows promise as a diagnostic technique for keratitis. We intend to expand the set of pathogens studied before assessing the feasibility of the technique in vivo by introducing cultures onto pig corneas.
*Department of Physics and Astronomy, University of Leicester, Leicester, United Kingdom; †Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom; and ‡Department of Ophthalmology, Leicester Royal Infirmary, Leicester, United Kingdom.
Philippa M
PM
Molyneux
Simon
S
Kilvington
Matthew J
MJ
Wakefield
Jeremy I
JI
Prydal
Nigel P
NP
Bannister
D015823:Acanthamoeba Keratitis / Q000175:diagnosis*; D048248:Acanthamoeba castellanii / Q000737:chemistry*; D000818:Animals; D001419:Bacteria / Q000737:chemistry; D015822:Eye Infections, Parasitic / Q000175:diagnosis*; D005658:Fungi / Q000737:chemistry; D006801:Humans; D061848:Optical Imaging; D025341:Principal Component Analysis / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*