Gram stain and addition of amphotericin B to improve the microbial safety of human donor corneas

Abstract

To determine the effectiveness of two methods to improve the microbial safety of human corneas preserved in organ culture. We compared the number of positive preservation solutions of corneas in organ culture in which the initial short-term hypothermic corneal maintenance solution was supplemented with amphotericin B 2.5 µg/mL and the historical data of microbial test results (2015–2019). In addition, we appraised the efficacy of Gram stain to detect bacterial or fungal contamination in the organ culture solutions of corneas from at-risk donors compared to the culture tests of corneas from not-at-risk donors. Statistical analysis was performed using STATA and statistical significance set at p < 0.05. The number of positive culture tests after preservation was 15 (0.5%) in 2020 compared to a mean of 37 (1.2%) in the period 2015–2019 (p < 0.01), with 10 (1.0%) positive samples in the cohort of 998 corneas from at-risk donors and 5 (0.2%) in the 2046 corneas from not-at-risk donors (p < 0.01). All corneas from at-risk donors tested positive at Gram stain and the results were available 1–3 days before those of the conventional culture tests. Amphotericin B supplementation in the short-term maintenance solution markedly reduced the number of positive microbial tests after organ culture and the early detection of contaminants, including slow-growing microorganisms, by Gram stain before the standard culture results. This meant fewer corneas being discarded and a greater likelihood of preventing post-graft infections.

Screening and optimization of a novel gallic acid and tannase production under semi quantitative and quantitative methods

“…Purified cultures were stored at 4 °C in brain heart infusion agar for short-term preservation [12], or stored at -20 °C in broth medium containing 20% glycerol for long-term preservation [13]. Gram staining was performed after incubation at 37 °C for 24 h [14]. Then biochemical (oxidase and catalase) assays were performed [15].…”

Ultrastructural Analysis of Rehydrated Human Donor Corneas After Air-Drying and Dissection by Femtosecond Laser

“…Sterility testing was achieved by sampling the Storage solution before the dehydration process, 6 days after the preservation in organ culture, and testing the Deswelling-Transport solution 24 h after the transfer of the dehydrated cornea prior to shipment to the surgical center. Microbial growth was screened in the media by means of two validated automated systems: Bactec 9240 (Becton-Dickinson, Franklin Lakes, NJ, USA) and HB&L (Alifax, Padua, Italy) (21).…”