Objective: This validation study investigates the treatment of cornea organ culture medium (Modified Eagle Medium, Biochrom GmbH, Berlin, Germany) with RESEP, a new medical device for antibiotics removal, before microbiological testing with BACTEC^TM blood culture bottles.

Methods and analysis: 10–100 colony forming units of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtillis, Aspergillus brasiliensis, Clostridium sporogenes, Enterobacter cloacae and Staphylococcus epidermidis were inoculated in 9mL of cornea organ culture medium.
In group A, the medium was withdrawn with RESEP and treated for 20 min at room temperature, and then inoculated in BACTEC Plus Aerobic/F/Anaerobic/F blood culture bottles.
In group B, the medium, spiked by the inoculation of microorganism, was injected directly. For each strain, a growth control was performed, by direct inoculation of the microorganisms in BACTEC^TM vials (positive control). All samples were incubated in the automated BACTEC^TM blood culture system at 36°C ±1°C for maximum of 14 days or until a positive reading. The elimination of antibiotics from the medium by RESEP was determined by high-performance liquid chromatography.

Results: After 20 min of RESEP treatment, 100% (n=9) of streptomycin, 100% (n=9) of amphotericin B and 99.7% (n=9) of penicillin G were eliminated.
In group A , all microorganisms were detected within 3 days of incubation with a sensitivity of 100% (n=99) and showed no significant delay compared with the positive controls.
In group B, the overall sensitivity was 67.9% (n=96) with a significant delay until detection of microbial growth for all tested microorganisms except for A. brasiliensis.

Conclusion: The use of RESEP to eliminate the antibiotics from cornea organ culture medium increases the sensitivity of the microbiological testing with BACTEC^TM Plus blood culture bottles significantly and fulfils the requirements of the European Pharmacopoeia method suitability test.

Purpose: To compare the protective properties and ease of manipulation during cataract surgery of corneal coating with a gel (eyeDRO; AL.CHI.MI.A. S.R.L, Italy) and corneal irrigation with balanced salt solution (BSS).

Methods: We analyzed the data of 51 patients receiving either eyeDRO or BSS during routine cataract surgery performed within a 20-day period in 2016. The selected parameters were intraoperative clarity and ease of manipulation; postoperative epithelial integrity; and patient discomfort.

Results: Compared with BSS irrigation, eyeDRO coating significantly increased intraoperative clarity and ease of manipulation (P < 0.01). Single application was required in eyeDRO-treated eyes, whereas BSS was applied 5.3 ± 0.4 times on average (P < 0.01). Two hours postoperatively, a normal epithelium was observed in 90.0% and 60.0% of eyeDRO-coated and BSS-irrigated eyes, respectively; punctate epithelial damage was observed in 9.7% and 40.0% (P < 0.05) of eyeDRO-coated and BSS-irrigated eyes, respectively; eye irritation and foreign body sensation were experienced by 13.0% and 37.0% of eyeDRO-treated patients and by 65.0% and 100% of BSS-treated patients, respectively (P < 0.01). Twenty-four hours postoperatively, 80.0% of BSS-treated patients versus 19.0% of eyeDRO-treated patients still experienced foreign body sensation (P < 0.01).

Conclusions: eyeDRO coating was shown to be a safer and more effective option than BSS irrigation in cataract surgery because single application provided optimal hydration and intraoperative clarity during the entire surgery, better preserved the corneal epithelium, and offered postoperative comfort to the patient.

Objective: This study aimed at validating the method for sterility testing of the corneal culture medium, TISSUE-C, and the transport/deswelling medium, CARRY-C, according to the method suitability test, as defined by the European Pharmacopoeia (EP), using RESEP, which is a new medical device for removal of antimicrobial agents and an automated culture system.

Methods and analysis: The six EP reference strains were inoculated in TISSUE-C and CARRY-C. Half of the samples were treated with RESEP (RESEP+ group) prior to the sterility testing, whereas the remaining samples were untreated (RESEP− group). Growth controls were obtained by direct inoculation of the micro-organisms in the culture broths. Microbial growth was read by an automated light scattering culture system within 48 hours.

Results: The use of RESEP allowed detection of microbial growth in 100% of the tested samples, with a mean time to detection (TTD) comparable with that of the growth control group. Significantly lower sensitivity (38.83%±20.03% for both media, P<0.05) and TTD variability, depending on the tested micro-organism, were observed in the RESEP− group. The method specificity was 100% for both groups.

Conclusion: The use of RESEP increased the sensitivity of the sterility testing method to 100% and, for the first time, allowed validation of the method for sterility testing of corneal storage media according to the EP method suitability test. This further increases the safety of the corneas intended for transplantation.

Abstract: We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives.

Abstract
The success of allotransplants is critically dependent on both tissue viability and efficient removal of potentially toxic cryopreservants. In this study we analysed the dimethyl sulphoxide (Me2SO) content of cardiovascular tissue samples stored in tissue banks and optimized a washing protocol to be used before surgical implant. We compared the Me2SO content of heart valves, arteries and veins and quantitatively determined by HPLC the washing kinetics of each group of tissue samples under strictly controlled conditions using an industrial washing medium(BASE).Our results showed that heart valves and arteries have significantly slower Me2SO release kinetics than veins. Approximately 20 % of the initial content of cryopreservant could still be detected in the valves and arterial tissue after 15 min
24 of continuous washing. Conversely, veins were almost completely cleared of the cryoprotectant under the same conditions. We propose a washing protocol consisting of two sequential washing with BASE for a total of 25 min for valves and arteries and 15 min for veins. In our hands, this protocol reliably ensures the removal of more than 95 % of the initial Me2SO content.

Abstract: The presence of residual antibiotics in tissue allografts after decontamination with antibiotic cocktails may result in widely documented adverse effects in predisposed subjects.

Moreover, antibiotic residues may mask contaminating microorganisms, resulting in falsely negative sterility tests, with potential risk of post-surgical infections. The objective of the present study was to define a rinsing procedure capable of eliminating antibiotic residues from cardiovascular, bone and skin tissues after decontamination with BASE.128. Different washing patterns, employing BASE medium, were applied.

The presence of antibiotic residues in tissue homogenates was assessed by agar diffusion test at different stages of tissue processing. To test whether antibiotic residues can result in falsely negative microbiological analysis, we induced a superficial tissue contamination with known inoculum concentration. By employing four different porcine tissues, we here report direct evidence that the presence of even limited amounts of antibiotics in decontaminated tissues interferes with sterility testing. This has implications in terms of increased risk of infections in allograft recipients.

To minimize this risk, we developed a procedure for extensive removal of antibiotics from allografts, allowing for subsequent detection of microbial contaminations that may occur during transportation, storage or processing prior to allograft transplantation. Our study emphasizes the importance of validating all processes and analytical methods in tissue banking, in order to warrant tissue safety.

This will minimize the risks of post-surgical infections as well as antibiotic-induced anaphylaxis in predisposed patients.