Detection of contamination during organ culture of the human cornea

Abstract

Corneas harvested post-mortem are at risk of contamination, therefore antibiotic additives are used in cold storage and organ culture systems. In the latter, sterility testing of the medium is part of the standard protocol. Intuitively, testing after longer organ culture periods should be more likely to detect contaminations than early testing, but may delay allocation. This study evaluates whether an optimal time for detection of donor cornea contamination can be identified. The study complies with the Declaration of Helsinki. All procedures were supervised using a certified quality management system (ISO 9001:2000). Donor corneas harvested by enucleation or corneoscleral excision over 5 consecutive years were processed according to German and EC laws and guidelines. The corneas were stored in a closed organ culture system in 100 ml MEM containing penicillin, streptomycin and amphotericin B at 31 degrees C for up to 28 days without media exchange. In 762 corneas, 10 ml samples of medium, obtained between days 3 and 8 of culture, were tested for sterility in an automated detection system (BacT/ALERT, bioMérieux). In 424 corneas, a second sterility test was performed from the same medium before release. Contamination detection probabilities were related to the culture duration before the primary test (Cochran-Armitage). Overall, 19 contaminations were found. Contaminations were bilateral in four donors. One contamination was apparent by macroscopic inspection of the medium prior to the primary sterility test; 12 were detected upon primary sterility testing. Furthermore, six primarily undetected contaminations were observed: five in the secondary sterility test and one suspected microscopically. In most cases, contamination could also be seen by medium turbidity and acidification, but in three cases macroscopic medium changes were significantly delayed or absent. No trends were found between the times of primary sterility sampling and both positive and false negative test outcome probabilities. Detection probability of contaminations in organ culture media does not increase between days 3 and 8; therefore, sterility can be tested on day 3. With the recommended follow-up after sterility testing being 7 days, microbiologic release can take place after 10 days of culture. Nevertheless, the testing is not failsafe, and should always be combined with macroscopic inspection of the media.