Measurements of antifungal levels in corneal tissue: a simplified bioassay for amphotericin B.

Measuring precise antifungal levels in the cornea with broth-dilution bioassays is difficult, as standard techniques involving visual determination of endpoints are hindered by corneal debris. To increase the precision of the measurement, we modified the sample preparation for bioassay of rabbit corneas treated with subconjunctival amphotericin B. Endpoint determination and variance were compared for a freshly thawed corneal suspension and the supernatant after 24 h equilibration; bioassay of the corneal suspension after 24 h equilibration served as an additional control. All endpoints were read visually in a masked fashion and were verified by culture. The three methods gave comparable endpoint values with equivalent degrees of variance. Amphotericin B levels were consistent by both visual and culture determination; however, endpoints were clearly visible and easier to read for the supernatant. Visual determination of the endpoints for the supernatant following 24 h equilibration simplified and ensured the precision of the bioassay technique.