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Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual.

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Presentation on theme: "Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual."— Presentation transcript:

1 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 1 Antibiotic cocktails and tissue processing: false negative results in microbiological analysis Laura Giurgola, Claudio Gatto, Jana D’Amato Tothova

2 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 2 Our previous studies showed that… The decontamination of tissue allografts with antibiotic cocktails can lead to an antibiotic carry-over effect, which in turn can result in false negative results in the microbiological analysis. (Gatto et al., EATB 2010)

3 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 3 Current microbiological tests METHODTARGETEVALUATION BACTEC ™ (BD)LiquidsCO 2 production BacT/ALERT® (Biomérieux)LiquidsCO 2 production HB&L ( ALIFAX) LiquidsTurbidity COLTURE MEDIA (Thyoglicollate/TSB) Liquids/TissuesTurbidity Methods based on bacterial growth What would happen in case of bacteriostasis? (state of suspended growth and/or reproduction of bacteria)

4 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 4 THE DEVICE REQUIRED THE PRODUCT STERILITY: -Difficulty to validate it’s sterility test due to the presence of antibiotic in the solution: possible false negative results - Development of a method for the removal of antibiotic residues Introduction We realized the problem of false negative results for the first time during the validation study of the BASE.128 medical device: decontamination solution for human tissues intended for transplantation containing vancomycin HCl, cefotaxime sodium salt, gentamicin sulphate, amphotericin B desoxycolate.

5 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 5 Interfering factor removal by ResEP device ResEP syringe: single-use device for elimination of antibiotics from liquid samples Tissue ResEP (prototype): single-use device for elimination of antibiotics from tissue samples

6 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 6 Purpose COMPARATIVE STUDIES WITH SOME ITALIAN TISSUE BANKS to evaluate currently used bacteriological analysis methods and the impact of false negative results in microbiological analysis Antibiotic cocktails are used in tissue processing : they could mask microbial growth in tissue and liquid samples undergoing microbiological tests

7 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 7 Organ culture for 21 days CORNEA procurement (n=33) SKIN procurement (n=20) CARDIOVASCULAR tissues procurement (n=47) 1. Microbiological Tissue Bank analysis 2. EP sterility test (ResEP device) 3. Antibiotic residue monitoring by HPLC Experimental design Decontamination, rinsing and cryopreservation

8 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 8 Materials and methods - SKIN AND CARDIOVASCULAR TISSUES: 3 bank-prepared antibiotic solutions or medical device BASE.128 (AL.CHI.MI.A.S.r.l.) - Cryopreservation 10% CRYO.ON (DMSO) in RPMI CORNEAS: cold and warm storage media Eusol-C, Tissue-C (AL.CHI.MI.A. S.r.l.) Tissue processing - STANDARD TISSUE BANK PROCEDURES: BACTEC ™(BD) method, BacT/ALERT®(Biomérieux) method and Thioglycollate/ TSB media. - AL.CHI.MIA. S.r.l. sterility test according to EP, after removal of antibiotic residues with ResEP device (AL.CHI.MI.A. S.r.l.). Microbiological analysis

9 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 9 Results: Antibiotic residue monitoring (HPLC) CARDIOVASCULAR TISSUES Cefotaxime Amphotericin B deoxycholate Vancomycin HCl Gentamicin sulfate TISSUE HOMOGENATES LIQUID SAMPLES Antibiotic concentration (µg/ml) Antibiotic concentration (µg per g of tissue)

10 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 10 Results: Antibiotic residue monitoring (HPLC) Cefotaxime Amphotericin B deoxycholate Vancomycin HCl Gentamicin sulfate SKIN TISSUES RINSING LIQUIDS TISSUE HOMOGENATES Antibiotic concentration (µg/ml) Antibiotic concentration (µg per g of tissue)

11 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 11 Antibiotic residues (µg/ml) LIQUID SAMPLES Results: Antibiotic residue monitoring (HPLC) CORNEAL TISSUES Amphotericin B deoxycholate Gentamicin sulphate Pennicilin G Streptomycin

12 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 12 Results: microbiological analysis CARDIOVASCULAR TISSUES NO REMOVAL OF ANTIBIOTIC RESIDUES (TB analysis) REMOVAL OF ANTIBIOTIC RESIDUES (EP test, ResEP device) positivenegativepositivenegative Cardiovascular (n=47) 2% (1/47) 98% (46/47) 19% (9/47) 81% (38/47)

13 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 13 Results: microbiological analysis SKIN TISSUES NO REMOVAL OF ANTIBIOTIC RESIDUES (TB analysis) REMOVAL OF ANTIBIOTIC RESIDUES (EP test, ResEP device) positivenegativepositivenegative Skin (n=20) 0% (0/20) 100% (20/20) 60% (12/20) 40% (8/20)

14 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 14 Results: microbiological analysis CORNEAL CULTURE MEDIUM NO REMOVAL OF ANTIBIOTIC RESIDUES (TB analysis) REMOVAL OF ANTIBIOTIC RESIDUES (EP test, ResEP device) positivenegativepositivenegative Cornea (n=33) 0% (0/33) 100% (33/33) 67% (22/33) 33% (11/33)

15 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 15 FALSE NEGATIVE RESULTS Cardiovascular17% Skin60% Cornea67% Results: microbiological analysis

16 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 16 Processing liquids (post decontamination and rinsing/cryopreservation solutions) and tissue samples submitted to sterility test showed important antibiotic content The use of ResEP device allowed the removal of antibiotic residues from the samples undergoing sterility tests thus increasing the reliability of the microbiological tests False negative results in microbiological tests are a common problem to all Tissue Banks (skin, cardiovascular and eye banks). Percentages vary depending on tissue, process, microbiological method, etc. The currently adopted microbiological systems do not assure the detection of false negative results, as they were not designed and/or validated to meet the requirements of the sterility test according to the Pharmacopoeia The complete removal of interfering factors from samples undergoing sterility tests is of paramount importance to obtain reliable results Conclusions

17 Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura Giurgola Polish Association for Tissue Banking 1 st Annual Scientific Meeting, Warsaw 17 Thank you for your attention! Research & Development Claudio Gatto Laura Giurgola Jana D’Amato Tothova Aknowledgements Pasqualepaolo Pagliaro Marina Buzzi Sabrina Manara Adriana Terzi Gianluca Polvani Luca Dainese Anna Guarino Barbara Micheli Raffaella Fasolo Sandra Rizzi Ilenia Mazzo Veronica Zenti Claudia Perbellini M Letizia Guastafierrro Aurora Vassanelli Tissue Bank of Verona A.Pocobelli C.Amici R.Donati Anna Limongelli Francesca Pateri Raffaela Mistò


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