Abstract
Abstract: :
Purpose:The occurrence of opportunistically pathogenic filamentous fungi and yeasts in the ocular tear film has been well documented. It has proved difficult however, to determine the biomass and activity of fungi in the ocular tear film and in the biofilm which forms over a contact lens. We report on the utilization of a technique to measure ergosterol as an indicator not only of the presence of fungi but also as a measure of fungal biomass. Methods:Thirteen subjects were enrolled in the study. A contact lens that had been worn continuously for periods of 1–2, 7, 14 or 30 days was removed from an eye of each subject, rinsed in sterile double distilled water and transferred to 100% methanol. In addition, a sterile, unworn control lens was treated in the same manner. Measured volumes were placed in 200 ml round bottom flasks along with an additional 15 ml of methanol per sample. Known quantities of pure ergosterol were added to replicate samples and used as a spike sample. Ergosterol extraction followed the methodology described by Newell and Fell ( 2001) and Schatz et al (2003). Lipids were extracted first using a reflux apparatus followed by sterol release and full separation by mixing with pentane and dry bath evaporation. Samples were analyzed via HPLC with a Perkin Elmer 785 A UV/VIS Absorbance Programmable Detector, Series 200 LC pump, Chromatography Interface 600 Series Link and PE Nelson 900 Series Interface. Injection standards were prepared with pure ergosterol in methanol with known values of 25 ug/ml, 50 ug/ml, and 100 ug/ml. All absorbance peaks were recorded at a wavelength of 282 nm. Results: The mean level of ergosterol was found to be 0.2487+/–0.258 ug/ml in lenses worn continuously for 1–2 days, 0.2833+/–0.283 ug/ml after 7 days, 0.3693 +/–0.272 ug/ml after 14 days and 0.4284 +/–0.094 in lenses worn for 30 continuous days. A considerable amount of interpatient variabilty was recorded. Ergosterol was not detected in the control lenses. Conclusions: The level of ergosterol is a useful measure of fungal biomass in the ocular tear film. The observed increase in fungal biomass on contact lenses over time is important in that certain cell wall constituents such as mannans are able to activate the alternative pathway of complement. It may be that, in some cases, fungi in the biofilm function as a catalyst for the occurrence of contact lens associated red eye (CLARE). Whether fungal adherence to contact lenses is material dependent, species dependent or subject dependent is not fully understood.
Keywords: contact lens • fungal disease • keratitis