Antibiotic cocktails and tissue processing: false negative results in microbiological analysis

1
Antibiotic cocktails and tissue processing false
negative results in microbiological analysis
Laura Giurgola, Claudio Gatto, Jana DAmato
Tothova
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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)

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Current microbiological tests
METHOD TARGET EVALUATION
BACTEC (BD) Liquids CO2 production
BacT/ALERT (Biomérieux) Liquids CO2 production
HBL (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)
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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 its 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

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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
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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
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Experimental design
CORNEA procurement (n33)
CARDIOVASCULAR tissues procurement (n47)
SKIN procurement (n20)
Organ culture for 21 days
Decontamination, rinsing and
cryopreservation
Decontamination,
rinsing and cryopreservation
1. Microbiological Tissue Bank
analysis
2.
EP sterility test (ResEP device)
3.
Antibiotic residue monitoring by HPLC
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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.).

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Results Antibiotic residue monitoring (HPLC)
LIQUID SAMPLES
CARDIOVASCULAR TISSUES
Vancomycin HCl Gentamicin sulfate
Cefotaxime Amphotericin B deoxycholate
Antibiotic concentration (µg/ml)
TISSUE HOMOGENATES
Antibiotic concentration (µg per g of tissue)
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Results Antibiotic residue monitoring (HPLC)
SKIN TISSUES
RINSING LIQUIDS
Vancomycin HCl Gentamicin sulfate
Cefotaxime Amphotericin B deoxycholate
Antibiotic concentration (µg/ml)
TISSUE HOMOGENATES
Antibiotic concentration (µg per g of tissue)
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Results Antibiotic residue monitoring (HPLC)
CORNEAL TISSUES
LIQUID SAMPLES
Antibiotic residues (µg/ml)
Pennicilin G Streptomycin
Amphotericin B deoxycholate Gentamicin sulphate
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Results microbiological analysis
CARDIOVASCULAR TISSUES CARDIOVASCULAR TISSUES CARDIOVASCULAR TISSUES CARDIOVASCULAR TISSUES CARDIOVASCULAR TISSUES
NO REMOVAL OF ANTIBIOTIC RESIDUES (TB analysis) NO REMOVAL OF ANTIBIOTIC RESIDUES (TB analysis) REMOVAL OF ANTIBIOTIC RESIDUES (EP test, ResEP device) REMOVAL OF ANTIBIOTIC RESIDUES (EP test, ResEP device)
positive negative positive negative
Cardiovascular (n47) 2 (1/47) 98 (46/47) 19 (9/47) 81 (38/47)
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Results microbiological analysis
SKIN TISSUES SKIN TISSUES SKIN TISSUES SKIN TISSUES SKIN TISSUES
NO REMOVAL OF ANTIBIOTIC RESIDUES (TB analysis) NO REMOVAL OF ANTIBIOTIC RESIDUES (TB analysis) REMOVAL OF ANTIBIOTIC RESIDUES (EP test, ResEP device) REMOVAL OF ANTIBIOTIC RESIDUES (EP test, ResEP device)
positive negative positive negative
Skin (n20) 0 (0/20) 100 (20/20) 60 (12/20) 40 (8/20)
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Results microbiological analysis
CORNEAL CULTURE MEDIUM CORNEAL CULTURE MEDIUM CORNEAL CULTURE MEDIUM CORNEAL CULTURE MEDIUM CORNEAL CULTURE MEDIUM
NO REMOVAL OF ANTIBIOTIC RESIDUES (TB analysis) NO REMOVAL OF ANTIBIOTIC RESIDUES (TB analysis) REMOVAL OF ANTIBIOTIC RESIDUES (EP test, ResEP device) REMOVAL OF ANTIBIOTIC RESIDUES (EP test, ResEP device)
positive negative positive negative
Cornea (n33) 0 (0/33) 100 (33/33) 67 (22/33) 33 (11/33)
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Results microbiological analysis
FALSE NEGATIVE RESULTS FALSE NEGATIVE RESULTS
Cardiovascular 17
Skin 60
Cornea 67
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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

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Aknowledgements
Gianluca Polvani Luca Dainese Anna
Guarino Barbara Micheli
Anna Limongelli Francesca Pateri Raffaela Mistò
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
A.Pocobelli C.Amici R.Donati
Claudio Gatto Laura Giurgola Jana DAmato Tothova
Research Development
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