jana d’amato tothova fate of antibiotics after the tissue decontamination phase m.sc. laura...
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Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st Annual Scientific Meeting, Warsaw
1
Antibiotic cocktails and tissue processing: false negative results in
microbiological analysis
Laura Giurgola, Claudio Gatto, Jana D’Amato Tothova
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st 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)
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st Annual Scientific Meeting, Warsaw
3
Current microbiological tests
METHOD TARGET EVALUATION
BACTEC ™ (BD) Liquids CO2 production
BacT/ALERT® (Biomérieux) Liquids CO2 production
HB&L (ALIFAX) Liquids Turbidity
COLTURE MEDIA(Thyoglicollate/TSB)
Liquids/Tissues Turbidity
Methods based on bacterial growth
What would happen in case of bacteriostasis?
(state of suspended growth and/or reproduction of bacteria)
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st 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.
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st 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
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st 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
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st 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
Decontamination, rinsing and
cryopreservation
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st 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 1640
- 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
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st Annual Scientific Meeting, Warsaw
9
Results: Antibiotic residue monitoring (HPLC)CARDIOVASCULAR TISSUES
0
20
40
60
80
100
120
Postdecontamination
liquid
Rinsing/cryopreservation
liquid
Postdecontamination
liquid
Rinsing/cryopreservation
liquid
NO ANTIBIOTIC REMOVAL ANTIBIOTIC REMOVAL
CefotaximeAmphotericin B deoxycholate
Vancomycin HClGentamicin sulfate
TISSUE HOMOGENATES
LIQUID SAMPLES
Antib
iotic
con
cent
ratio
n (µ
g/m
l)An
tibio
tic c
once
ntra
tion
(µg
per g
of ti
ssue
)
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st Annual Scientific Meeting, Warsaw
10
Results: Antibiotic residue monitoring (HPLC)
CefotaximeAmphotericin B deoxycholate
Vancomycin HClGentamicin sulfate
SKIN TISSUESRINSING LIQUIDS
TISSUE HOMOGENATES
Antib
iotic
con
cent
ratio
n (µ
g/m
l)An
tibio
tic c
once
ntra
tion
(µg
per g
of ti
ssue
)
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st Annual Scientific Meeting, Warsaw
11
LIQUID SAMPLES
Results: Antibiotic residue monitoring (HPLC)CORNEAL TISSUES
Amphotericin B deoxycholateGentamicin sulphate
Pennicilin GStreptomycin
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st 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)
positive negative positive negative
Cardiovascular (n=47)
2%(1/47)
98%(46/47)
19%(9/47)
81%(38/47)
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st 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)
positive negative positive negative
Skin (n=20)
0%(0/20)
100%(20/20)
60%(12/20)
40%(8/20)
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st 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)
positive negative positive negative
Cornea (n=33)
0%(0/33)
100%(33/33)
67% (22/33)
33% (11/33)
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st Annual Scientific Meeting, Warsaw
15
FALSE NEGATIVE RESULTS
Cardiovascular 17%Skin 60%Cornea 67%
Results: microbiological analysis
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st 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
Jana D’Amato Tothova
Fate of antibiotics after the tissue decontamination phase
M.Sc. Laura Giurgola
Polish Association for Tissue Banking1st Annual Scientific Meeting, Warsaw
17Thank you for your attention!
Research & Development
Claudio GattoLaura GiurgolaJana D’Amato Tothova
Aknowledgements
Pasqualepaolo PagliaroMarina BuzziSabrina ManaraAdriana Terzi
Gianluca PolvaniLuca DaineseAnna GuarinoBarbara Micheli
Raffaella FasoloSandra RizziIlenia MazzoVeronica ZentiClaudia PerbelliniM Letizia GuastafierrroAurora Vassanelli
Tissue Bank of VeronaA.PocobelliC.AmiciR.Donati
Anna Limongelli Francesca Pateri Raffaela Mistò