Utility of the Limulus Amoebocyte Lystate(LAL) to Detect Fungal Contamination in Contact Lense Cases

Abstract

Purpose:: The Limulus amoeba lystate (LAL) has been used to detect endotoxins both in nonocular and ocular tissues. Recently, the LAL has been modified to take advantage of its ability to also react with 1,3 D-glucan present in fungal cell walls. We evaluate the utility of the LAL gel clot assay to detect fungal cell wall contamination of patientsâ€^TM contact lens cases

Methods:: In the first half of the study, we first evaluated the sensitivity of the LAL to detect contamination of three commercial contact lens solutions (Renu Moisture Loc, Opti Free, and Complete). Each solution was diluted 1:10 and inoculated with aliquots of 103 (low) and 106 (high) cfu/ml of Fusarium oxysporum isolated from the contact lens case of a patient during the recent fusarium keratitis outbreak. 200 ul of each commercial solution times 3 (1:10 dilution only, 1:10 with 103 cfu/ml and 1:10 with 106 cfu/ml) was inoculated into individual single test vials of LAL with a detection limit of 0.125 units of endotoxins. Each of the aliquots was also inoculated into a positive control LAL test vial to rule out interfering substances. Positive and negative controls were also included. All tubes were incubated at 35 C for 1 hour. A positive result was indicated by a firm clot that did not dissolve upon inversion, an equivocal or weakly positive result was one in which the clot dissolved upon inversion and a negative rest was indicated by the absence of a clot. In the second phase, eleven patient contact lens cases collected from March 2006 â€"October 2006 were randomly chosen for evaluation. Three of the eleven patients grew Fusarium species from initial cultures; the remainder was positive for gram negative rods. Solution from each was diluted 1:10 and evaluated as above.

Results:: A firm clot was documented for both the low and high fusarium concentrations for all three commercial solutions. The presence of interfering substances was not detected at the 1:10 dilution. All 11 patient samples were weakly positive for the presence of endotoxins/1,3 glucan.

Conclusions:: The LAL could play a role in detection of both endotoxin and 1,3 glucan contamination of contact solutions/cases. Further refinement of using LAL to detect fungal cell wall contamination may be necessary.

Keywords: clinical laboratory testing • keratitis