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.

Summary: A spanish study by electronical microscopy on corneas for DMEK, stored in Tissue-C at 31°C, shows that there are morphological and structural differences between the center and the periphery of the grafted tissue.

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.

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: 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.

Background: Antibiotics can interfere with microbial growth during microbiological analysis and lead to false negatives. The aim of the study was to validate the RESEP syringe 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.

Materials and methods: The elimination of Streptomycin, Penicillin G, Gentamicin, Cefotaxime, Vancomycin, Amphotericin B deoxycholate from 3-9 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 syringe.
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 syringe. The tissue bank operators treated 20 samples of cold storage media with the RESEP syringe in order to evaluate the ease of use, operation time, volume compatibilities and time to detection of automated systems (Bactec BD, BactAlert Biomérieux and H&B Alifax).

Results: Before RESEP syringe 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 syringe.
Microbial recovery of 1-10 and 10-100 CFU was obtained for all investigated strains and from all the tested media. The RESEP syringe was compatible with tissue bank processes and all the evaluated microbiological systems. The RESEP syringe treated samples reduced significantly the time to detection and resulted in additional positive samples when compared to by automated systems without using the RESEP syringe.

Conclusions: The RESEP syringe 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 syringe 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.
The study performed by two Italian Eye Banks and AL.CHI.MI.A.’s R&D Dept. on sixty-one corneal tissues unsuitable for transplantation.
The corneal tissues retrieved by the personnel of the eye banks were transported to the bank in Eusol-C (AL.CHI.MI.A. Srl, Italy), transferred to Tissue-C (AL.CHI.MI.A. Srl, Italy) and stored for 12-14 days at 31°C.
Microbiological analyses were performed post-processing on Tissue-C by the eye banks, according to bank standard procedures using BACTEC plus aerobic/anaerobic system (BD); in parallel, microbiological analyses 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).
Sterility test after removal of interfering antibiotics with RESEP showed 59% of positive (contaminated) samples on average at the end of the process; the percentage of positive results varied depending on the bank. 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.
The presence of contaminants in the media at the end of the storage process indicates that standard corneal storage media does not guarantee efficient corneal decontamination.

Purpose: To evaluate the feasibility and outcome of combined Descemet’s membrane endothelial keratoplasty (DMEK), phacoemulsification and intraocular lens (IOL) implantation in the management of concomitant endothelial disorder, shallow chamber and cataract.

Setting: Clinical study at a tertiary referral center.

Methods: Prospective, nonrandomized study that included eight eyes of eight patients with concomitant endothelial disease and advanced cataract. In a first group of eight consecutive eyes, combined phacoemulsification, IOL implantation and DMEK wereperformed. Grafts were inserted with an ICL injector, encapsulated within viscoelastic plugs. Femtosecond laser-assisted descemetorhexis was performed in one case. The main
outcome measures were best spectacle corrected visual acuity (BSCVA), endothelial cell density (ECD) and intra- and postoperative complications.

Results: The procedure was successful in 75% (6/8) of cases. Two required a secondary regraft due to complete graft detachment and late failure. At six months, mean ECD loss was 55.4±24.0%, with an absolute mean ECD of 1248.3±755.0 cells/mm2. At six months, 100% (5/5) of patients with good visual potential reached BSCVA of ≥0.5, 80% (4/5) ≥0.8, and 60% (3/5) ≥1.0.
CONCLUSIONS: The combined triple procedure seems to be safe and effective in the management of cases with concomitant advanced Fuchs’ endothelial dystrophy, advanced cataract and shallow chamber. Endothelial cell density loss beyond six months remains under investigation.

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.
Sixty-one corneal tissues unsuitable for transplantation were retrieved by the personnel of two Italian eye banks and transported to the bank in Eusol-C (AL.CHI.MI.A. Srl, Italy); After transfer to Tissue-C (AL.CHI.MI.A. Srl, Italy), corneas were stored for 12-14 days at 31°C.
Microbiological analyses were performed post-processing on Tissue-C by the eye banks, according to bank standard procedures using BACTEC plus aerobic/anaerobic system (BD); in parallel, microbiological analyses were performed by AL.CHI.MI.A. Srl with sterility test according to the European Pharmacopeia, after removing potential interfering antibiotics with RESEP (AL.CHI.MI.A. Srl, Italy).
Sterility test after removing potential interfering antibiotics with RESEP showed an average of 59% positive samples at the end of the process; the percentage of positive results varied depending on the Bank. None of these contaminations was detected by BACTEC.
Removal of antibiotic residues from corneal storage media with the RESEP device resulted in a significant number of false negative results in the microbiological analyses.
The presence of contaminants in the media at the end of the storage process indicates that standard corneal storage media does not warrant efficient corneal decontamination.