Purpose: This study aimed at validating the method for sterility testing of the corneal culture medium, TISSUE-C (AL.CHI.MI.A. S.r.l.), and the transport/deswelling medium, CARRY-C (AL.CHI.MI.A. S.r.l.), according to the method suitability test, as defined by the European Pharmacopoeia (EP), using RESEP (AL.CHI.MI.A. S.r.l), which is a new medical device for removal of antimicrobial agents, and HB&L (Alifax) automated culture system.

Materials and Methods: 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 microorganisms in the culture broths. Microbial growth was read by HB&L automated light scattering culture system within 48 h.

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 microorganism, 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.

Purpose: The aim of this study was to validate the method for sterility testing of corneal storage media Tissue-C and Carry-C according to the “Method suitability test” (EP) using BACTEC (Becton Dickinson) automated system in a multicentric study.

Material & Methods: The validation study was performed at the Eye Bank of Rome and Eye Bank of Monza, Italy. Samples of organ culture medium (Tissue-C, AL.CHI.MI.A. S.r.l.), deswelling/transport medium (Carry-C, AL.CHI.MI.A. S.r.l.), and optimal growth media (growth control) were inoculated with 6 EP reference strains to obtain final microbial concentration of 10 cfu/ml, and tested at least in triplicate with BACTEC automatized system.
Method sensitivity, specificity and robustness were determined for each medium, with and without antibiotic removal from samples with RESEP (AL.CHI.MI.A. S.r.l.).

Results: Both eye banks obtained the same method sensitivity and specificity results. The method for sterility testing of Tissue-C and Carry-C samples after RESEP-treatment using BACTEC system showed 100% sensitivity and specificity. Samples treated with RESEP showed similar times to detection as compared to growth controls.

Conclusions: BACTEC system can be considered validated with 100% sensitivity and specificity, and robustness for samples of corneal storage media contaminated with 1-10 cfu/ml, and treated with RESEP.

Purpose: This study aimed at assessing the antimycotic activity of the new cold storage medium, Kerasave, and at evaluating the quality of donor corneas preserved in the medium at 4°C for 14 days in comparison with Optisol GS.

Material and Methods: Kerasave antimycotic activity was determined by in vitro time-kill studies using sterile porcine corneal tissues, contaminated with 10⁴ cfu/ml of C. Albicans (ATCC10231 and clinical isolate). The killing rate of the microorganisms was monitored at 4°C after 5 and 10 days of incubation in Kerasave.

Kerasave performance was assessed on 16 pairs of human corneas not suitable for transplantation, procured and evaluated according to standard procedures of Monza Eye Bank, Italy. One cornea was transferred in Kerasave and the contralateral in Optisol GS. Endothelial cell density (ECD), measured by specular microscopy (Keratoanalyzer, Konan), was evaluated pre-processing, and after 7 and 14 days of storage at 4°C. Endothelial cell morphology and mortality were determined according to Stocker method, and epithelial integrity, and corneal transparency were evaluated using a Slit lamp.

Results: In vitro time-kill studies showed a 3 to 4 log₁₀ reduction for both Candida strains within 10 days of incubation at 4°C.
Kerasave- and Optisol-GS-treated tissues showed similar ECD, mortality and endothelial morphology after 7 and 14 days of cold storage. Slit lamp analysis showed comparable corneal transparency and epithelial integrity in both groups.

Conclusions: The new cold storage medium with antimycotic tablet, Kerasave, exhibited an excellent antimycotic activity and biocompatibility with donor corneas after corneal storage at 4°C for up to 14 days.

Abstract: The aim of this study was to validate the microbiological testing of cornea organ culture medium according to “Method Suitability Test” defined by the European Pharmacopoeia, using blood culture bottles and the automated BACTEC system (BD), after removal of antibiotics with the new medical device RESEP (AL.CHI.MI.A. Srl, Italy).
10-100 CFU of Staphylococcus Aureus, Pseudomonas Aeruginosa, Candida Albicans, Bacillus Subtilis, Aspergillus Niger, Clostridium Sporogenes, Enterobacter Cloacae and Staphylococcus Epidermidis were inoculated in 9 ml cornea organ culture medium (MEM with 2% fetal calf serum and Streptomycin (130 µg/ml), Penicillin (60 µg/ml) and Amphotericin B (2,5 µg/ml), Biochrom). The medium was treated with RESEP for 20 minutes, inoculated in BACTEC plus vials (BD) and incubated until a positive reading or for at least 14 days at 36 ± 1°C. Untreated medium, positive and negative control samples were also tested. The elimination of antibiotics from the medium by RESEP was determined by HPLC.
The antibiotics were completely eliminated after 20 min. of treatment with RESEP at room temperature. In RESEP treated samples, the growth of all microorganisms was detected within 3 days, and showed no delay compared to the positive control samples. In contrast, the untreated medium samples showed no repeatable results of Staphylococcus Aureus, Enterobacter Cloacae, Candida Albicans and Bacillus Subtilis.
The microbiological testing of cornea organ culture medium with the automated BACTEC blood culture system using RESEP for the removal of antibiotics, could be successfully validated according to the “Method Suitability Test” of the European Pharmacopoeia (chapter 2.6.1.).

Purpose: aim of the study was to compare the direct inoculum method of corneal preservation media in the presence of antibiotics with the same method after elimination of antibiotics with the RESEP device.

Methods: 42 samples of cold storage media (EUSOL-C, AL.CHI.MI.A. S.r.l.) were obtained from the Eye Bank of Rome after donor cornea storage for up to 7 days. Aliquots of 3 ml of the media were either inoculated directly in trypton soy broth and fluid thioglycollate medium or treated with RESEP to eliminate antibiotic residues and then inoculated in growth media. 10 samples of sterile broth, treated with RESEP and tested by direct inoculum method, served as controls. To evaluate the initial contamination, bioburden was performed for each of the tested EUSOL-C by membrane filtration (MILLIFLEX SENSOR II – MILLIPORE). Positive samples were identified genetically.

Results: 19 out of 42 tested samples (45.2%) resulted contaminated when assessed by the direct inoculum method without RESEP treatment. The same samples treated with RESEP showed 25 of 42 (59.5%) positive samples. The RESEP positive samples, which were not detected without antibiotic elimination, were contaminated with Staphylococcus spp, Bacillus spp, Propionibacterium acnes. Turbidity was detected up to 5 days in advance using RESEP. None of the control samples was contaminated.

Conclusions: The elimination of antibiotic residues from samples allowed to reveal an additional 14% of contaminated samples as compared to the direct inoculum method without elimination of antibiotics. Our study demonstrated that the antibiotics present in the corneal preservation media may interfere with the microbial growth during microbiological analyses by direct inoculum method and lead to false negatives.

Abstract: Antibiotics can interfere with microbial growth during microbiological analysis and lead to false negatives. The aim of the study was to validate the RESEP device in terms of elimination of antibiotics and recovery of microorganisms from samples undergoing microbiological analysis and to evaluate its compatibility with automated microbiology systems and tissue bank processes.
The elimination of Streptomycin, Penicillin G, Gentamicin, Cefotaxime, Vancomycin, Amphotericin B deoxycholate from 3-6 ml of different corneal preservation media (EUSOL-C, TISSUE-C and CARRY-C) and a tissue decontamination medium (BASE.128) was determined by HPLC after 20 minutes of treatment with the RESEP device.
The recovery of living microorganisms from 3 ml of antibiotic media inoculated with 1-10 and 10-100 CFU of S. aureus, P. aeruginosa, C. albicans, B. subtilis, A. niger and C. sporogenes was determined by dilution plating method after 20 minutes treatment with the RESEP device. The tissue bank operators treated 20 samples of cold storage media with the RESEP in order to evaluate the ease of use, operation time, volume compatibilities and time to detection of automated systems (BACTEC, BD; Bact/ALERT, Biomérieux and H&B Alifax).
Before RESEP treatment, the concentration of antibiotics (Streptomycin, Penicillin G, Gentamicin, Cefotaxime, Vancomycin) in the tested media ranged from 110 to 140 μg/ml. Amphotericin B deoxycholate concentration was approximately 20 μg/ml. For all the tested media, total elimination of antibiotics was observed after 20 minutes of treatment with the RESEP device.
Microbial recovery of 1-10 and 10-100 CFU was obtained for all investigated strains and from all the tested media. The RESEP device was compatible with tissue bank processes and all the evaluated microbiological systems. The RESEP treated samples reduced significantly the time to detection and resulted in additional positive samples when compared to by automated systems without using the RESEP device.
The RESEP device was validated for elimination of antibiotics from all tested media and recovery of 1-10 and 10-100 CFU from tested samples. The simple and fast use of the RESEP was compatible with the tissue bank processes and allowed to enhance the performance of the automated microbiology systems used by the tissue banks.

Introduction: Corneal culture media contain antibiotics that might interfere with microbial growth during microbiological analysis and consequently result in false negatives.

Purpose: The aim of the study was to investigate the effects of antibiotic residues on microbiological analyses of donor corneas after culture in eye bank.

Material and methods: Sixty-one corneal tissues retrieved by the personnel of the eye banks were transported to the bank in EUSOL-C (AL.CHI.MI.A. S.r.l., Italy) and evaluated according to standard eye bank procedures. The tissues were then transferred to TISSUE-C (AL.CHI.MI.A. S.r.l., Italy) for tissue culture at 31°C for 12-14 days.
Microbiological analyses of the culture media were performed by the eye banks at the end of the culture, using BacTEC plus aerobic/anaerobic system (BD, Germany), according to bank standard procedures. The same culture media were sampled and analysed by R&D AL.CHI.MI.A using the RESEP tube device (AL.CHI.MI.A. S.r.l, Italy) that allows to remove the antibiotics from samples undergoing the test.

Results: Microbiological analyses with the RESEP tube showed that 59% of samples were contaminated on average. The percentage of positive results varied depending on the eye bank. None of these contaminations was detected by the BacTEC system.

Conclusions: Removal of antibiotic residues from corneal storage media with the ResEP tube resulted in a significant number of false negatives in the microbiological analyses.
The presence of contaminants in the media at the end of the storage process indicates that standard corneal storage media do not guarantee efficient corneal decontamination.

Abstract: The use of antibiotic cocktails during corneal processing can lead to an antibiotic carry-over effect, which in turn can generate false negative results in microbiological analysis.
The aim of the study was to investigate the impact of antibiotic residues on microbiological analyses of organ cultured donor corneas.
Twenty-four corneal tissues were retrieved by the personnel of the Eye Bank of Monza (Italy) and transported to the bank in Eusol-C (AL.CHI.MI.A. Srl, Italy). Tissues were transferred to Tissue-C (AL.CHI.MI.A. Srl, Italy), stored for 12-14 days at 31°C and then placed in the deswelling/transport medium Carry-C (AL.CHI.MI.A. Srl, Italy) for 24 h at room temperature.
Microbiological analyses were performed pre-processing on Eusol-C and post-processing on Tissue-C and Carry-C by the Eye Bank of Monza, according to bank standard procedures using BACTEC plus aerobic/anaerobic; in parallel, microbiological analysis were performed by AL.CHI.MI.A. with sterility test according to the European Pharmacopeia, after removing potential interfering antibiotics with RESEP (AL.CHI.MI.A. Srl, Italy).
Pre-processing microbiological analysis of the media, showed that 75% of the samples were contaminated (Staphylococcus spp.). 25% of such contaminations were not detected by BACTEC, thus yielding false negative results.
45% of the samples remained positive at the end of the process. None of these contaminations was detected by BACTEC.
Removal of antibiotic residues from corneal storage media with RESEP resulted in a significant number of false negative results in the microbiological analyses of the media.
The presence of contaminants in the media at the end of the storage process indicates that standard corneal storage media do not guarantee efficient decontamination of donor corneas.

Abstract: The aim of the study was to evaluate two strategies for decontamination of donor cornea.
Thirty donor corneas were procured by Monza Eye Bank. Ten corneas were transferred into decontamination medium “A” at 31°C for 28 days. Twenty corneas were decontaminated with medium “B” at 22°C, overnight, and subsequently stored either in Eusol-C (AL.CHI.MI.A. S.r.l) at 4°C for 10 days or in Tissue-C (AL.CHI.MI.A. S.r.l) at 31°C for 20 days. The decontamination phase was skipped for control tissues, which were stored either in Eusol-C or in Tissue-C. Microbiological contamination, endothelial cell density (ECD), endothelial morphology and mortality were monitored pre-processing, 24h post-decontamination and post-processing. For microbiological analyses, both Bactec system (BD) and sterility test according to European Pharmacopeia after antibiotic residues removal were used. Presence of antibiotic residues was monitored by HPLC.
Almost all tissues were initially contaminated with bacterial and/or fungal species. All post-decontamination corneas showed negative microbiological analyses. In both control and post-decontamination samples, HPLC analysis showed presence of antibiotics prior to microbiological analyses. Corneas stored in decontamination solution “A” did not show variation of ECD or endothelial morphology up to 14 days. After 21 days, endothelial morphology was severely altered as compared to control tissues. Overnight decontamination with medium “B” resulted in similar ECD, endothelial morphology and mortality rate as compared to control tissues.
Overnight decontamination, before tissue conservation, allowed to eliminate all contaminants from donor corneas without tissue alteration. Presence of antibiotic residues could interfere with microbial growth. Therefore their accurate removal is necessary prior to microbiological testing.

Abstract: A comparative analysis between Eusol-C and Borzenok-Moroz medium revealed a positive comparison of clinical and laboratory data between the two media.