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.

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: To explore the utility of multimodal microscopy as a noninvasive tool to assess corneal collagen cross-linking (CXL) efficacy, we investigated the correlation between riboflavin (RF) axial profile, second harmonic generation (SHG) imaging, and histological/biochemical changes of human corneas after RF-ultraviolet A (UVA)-catalyzed CXL.De-epithelialized human corneoscleral tissues were imaged by confocal and multiphoton microscopy to study RF tissue diffusion profile and SHG-based roughness index (Rq) after CXL. We installed 0.1% RF for 5, 10, and 20 minutes, respectively, followed by UVA irradiation, while dextran drug vehicle-treated corneas served as controls. Masson’s trichrome staining and collagenase digestion assay were employed to assess ultrastructural modifications of collagen lamellae and bioenzymatic strength following RF-UVA CXL.Stromal absorption of RF was significantly higher in 20 minutes compared with 5- and 10-minute drug instillations. The roughness index of SHG images was reduced after RF-UVA CXL at all RF instillation time points compared with dextran controls. Interestingly, correlation between axial profiles of RF dosage and Rq index was only observed in 10- and 20-minute RF instillations (R(2) = 0.13 and 0.28, respectively, all P < 0.05), but not in the 5-minute group. Masson’s trichrome staining revealed collagen fibril compaction in cross-linked corneas in an RF dose-dependent manner. Collagenase digestion assay showed significantly increased biochemical strength by higher RF doses in cross-linked corneas.Intrastromal RF distribution profiles correlated with histological and functional property changes in RF-UVA cross-linked corneas. A riboflavin-defined threshold further determined the sensitivity of SHG imaging as a noninvasive imaging modality to assess the efficacy of RF-UVA CXL.

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.