Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura...

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





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

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)





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

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

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

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





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





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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

)





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

)





11

LIQUID SAMPLES

Results: Antibiotic residue monitoring (HPLC)CORNEAL TISSUES

Amphotericin B deoxycholateGentamicin sulphate

Pennicilin GStreptomycin





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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)





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SKIN TISSUES


(TB analysis)




Skin (n=20)

0%(0/20)

100%(20/20)

60%(12/20)

40%(8/20)





14


CORNEAL CULTURE MEDIUM


(TB analysis)




Cornea (n=33)

0%(0/33)

100%(33/33)

67% (22/33)

33% (11/33)





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FALSE NEGATIVE RESULTS

Cardiovascular 17%Skin 60%Cornea 67%






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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





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

Jana D’Amato Tothova Fate of antibiotics after the tissue decontamination phase M.Sc. Laura...

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