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S. Schatz, K. Adams, H. Laubach; The Detection of Ergosterol on Contact Lenses and in the Contact Lens Biofilm . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3683.
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Purpose: The chemical components of the ocular tear film and the organisms which inhabit the ocular tear film have been studied in detail. However, little notice has been given to the indigenous filamentous fungi and yeasts that inhabit the ocular tear film. We report on a technique to measure ergosterol as an indicator for the presence of fungi in the biofilm that develops over and within a hydrogel contact lens. Methods: Hydrogel contact lenses that had been worn continuously for periods of two and four weeks were removed from the eye, 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 into 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 (2000). Using a reflux apparatus lipids were extracted first followed by sterol release and fully separated after 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 analysis of the contact lens biofilm using HPLC allowed us to measure microgram amounts of ergosterol that had been extracted from worn contact lenses. Ergosterol was detected at a level of 0.2524 ug/ml in the lenses that had been worn for two continuous weeks without disinfection and 0.3741 ug/ml in the lenses that had been worn continuously for four weeks. Ergosterol was not detected in the control lenses. Conclusions: While the presence of fungi in the ocular tear film is known this is the first report on the application of a method to quantify not only the amount of fungi present in the contact lens bio-film but also as a way to measure the level of fungal activity. Certain cell wall constituents such as mannans are able to activate the alternative pathway of complement. The trigger for corneal keratitis and corneal ulceration secondary to contact lens wear or overwear has largely been attributed to a bacterial etiology. It may be, in some cases, that an undue build up of yeasts in the contact lens bio-film may be a causative factor in corneal inflammation and ulceration.
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