Antibiotic cocktails and tissue
processing: false negative results in
microbiological analysis
Laura Giurgola, Claudio Gatto, Jana D’Amato Tothova
1
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
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)
2
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
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)
3
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
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.
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
4
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
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
5
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
Purpose
Antibiotic cocktails are used in tissue processing :
they could mask microbial growth in tissue and liquid samples undergoing
microbiological tests
COMPARATIVE STUDIES WITH SOME ITALIAN TISSUE BANKS
to evaluate currently used bacteriological analysis methods and the
impact of false negative results in microbiological analysis
6
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
Experimental design
CORNEA procurement
(n=33)
SKIN procurement
(n=20)
CARDIOVASCULAR tissues
procurement (n=47)
Organ culture for 21 days
Decontamination,
rinsing and
cryopreservation
Decontamination,
rinsing and
cryopreservation
1.
Microbiological
Tissue Bank analysis
7
2.
EP sterility test
(ResEP device)
3.
Antibiotic residue
monitoring by HPLC
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
Materials and methods
Tissue processing
- 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.)
Microbiological analysis
- 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.).
8
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
Results: Antibiotic residue monitoring (HPLC)
LIQUID SAMPLES
CARDIOVASCULAR TISSUES
Antibiotic concentration (µg/ml)
120
Vancomycin HCl
Gentamicin sulfate
Cefotaxime
Amphotericin B deoxycholate
100
80
60
40
20
0
Post
decontamination
liquid
Rinsing/
cryopreservation
liquid
NO ANTIBIOTIC REMOVAL
Post
decontamination
liquid
Rinsing/
cryopreservation
liquid
ANTIBIOTIC REMOVAL
TISSUE HOMOGENATES
Antibiotic concentration
(µg per g of tissue)
120
100
80
60
40
20
0
9
NO ANTIBIOTIC REMOVAL
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
ANTIBIOTIC REMOVAL
Results: Antibiotic residue monitoring (HPLC)
SKIN TISSUES
Antibiotic concentration (µg/ml)
RINSING LIQUIDS
Vancomycin HCl
Gentamicin sulfate
16
14
12
Cefotaxime
Amphotericin B deoxycholate
10
8
6
4
2
0
No antibiotic removal
Antibiotic removal
Antibiotic concentration
(µg per g of tissue)
TISSUE HOMOGENATES
10
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
Results: Antibiotic residue monitoring (HPLC)
CORNEAL TISSUES
LIQUID SAMPLES
160
Antibiotic residues (µg/ml)
140
120
100
80
60
40
20
0
no antibiotic
removal
antibiotic removal
TRANSPORT AND COLD STORAGE
MEDIUM
Pennicilin G
Streptomycin
11
no antibiotic
removal
antibiotic removal
ORGAN CULTURE MEDIUM
Amphotericin B deoxycholate
Gentamicin sulphate
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
Results: microbiological analysis
CARDIOVASCULAR TISSUES
NO REMOVAL
OF ANTIBIOTIC RESIDUES
(TB analysis)
Cardiovascular
(n=47)
REMOVAL
OF ANTIBIOTIC RESIDUES
(EP test, ResEP device)
positive
negative
positive
negative
2%
(1/47)
98%
(46/47)
19%
(9/47)
81%
(38/47)
12
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
Results: microbiological analysis
SKIN TISSUES
NO REMOVAL
OF ANTIBIOTIC RESIDUES
(TB analysis)
Skin
(n=20)
REMOVAL
OF ANTIBIOTIC RESIDUES
(EP test, ResEP device)
positive
negative
positive
negative
0%
(0/20)
100%
(20/20)
60%
(12/20)
40%
(8/20)
13
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
Results: microbiological analysis
CORNEAL CULTURE MEDIUM
NO REMOVAL
OF ANTIBIOTIC RESIDUES
(TB analysis)
Cornea
(n=33)
REMOVAL
OF ANTIBIOTIC RESIDUES
(EP test, ResEP device)
positive
negative
positive
negative
0%
(0/33)
100%
(33/33)
67%
(22/33)
33%
(11/33)
14
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
Results: microbiological analysis
FALSE NEGATIVE RESULTS
Cardiovascular
Skin
Cornea
15
17%
60%
67%
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
Conclusions
 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
16
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
Aknowledgements
Gianluca Polvani
Luca Dainese
Anna Guarino
Barbara Micheli
Pasqualepaolo Pagliaro
Marina Buzzi
Sabrina Manara
Adriana Terzi
Tissue Bank of Verona
Raffaella Fasolo
Sandra Rizzi
Ilenia Mazzo
Veronica Zenti
Claudia Perbellini
M Letizia Guastafierrro
Aurora Vassanelli
Anna Limongelli
Francesca Pateri
Raffaela Mistò
A.Pocobelli
C.Amici
R.Donati
Claudio Gatto
Laura Giurgola
Research & Development Jana D’Amato Tothova
Polish
for Tissue Banking
Fate of antibiotics after
theAssociation
tissue decontamination
phase
st
1 Annual Scientific Meeting, Warsaw
Jana
M.Sc.
D’Amato
Laura Giurgola
Tothova
Thank you for your attention!
17