towards rapid detection of staphylococcus aureus during blood culture world congress and expo on...
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Towards rapid detection of Staphylococcus aureus during blood culture
World Congress and Expo on Applied MicrobiologyAugust 18-20, 2015, Frankfurt
Vincent Templier, PhD StudentCEA, INAC-SPrAM-CREAB, F-38000 Grenoble,
France
Contact : [email protected]
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The threat of bacteremia
2
Intr
oduc
tion
• Bacteremia or bloodstream infection (BSI) = presence of viable bacteria in blood1,2.
• Affects mainly immunocompromized patients but not only.
• 200 000-250 000 cases / year in the USA
• Mortality can be as high as 20-50%3,4.
• S. aureus = major pathogen, accounting for almost 1/5 of bacteria involved in BSI5.
Bacteremia = life-threatening infection which needs rapid medical care.
1Reimer, L.G. et al., Clin Microbiol Rev, 1997 ; 2Wilson, M.L. et al., Clinical and Laboratory Standards Institute, 2007 ; 3Bearman, G.L., Archive of Medical Research, 2005 ; 4Dellinger, R.P. et al., Critical Care Medicine, 2013 ; 5Timsit, J.F., et al., BMC Infect Dis, 2014.
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Current procedure for microbial identification
3
Intr
oduc
tion
Hemoculture
12-36h
Empirical antibiotic treatment
Patie
nt
Bloo
d sa
mpl
e
(5 to
10m
L)
Appropriate
dilution
Low contamination
(1 CFU / 10mL)
1. Assessment of bacterial presence
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Current procedure for microbial identification
4
Intr
oduc
tion
Hemoculture
12-36h
Gram coloration and
Microscopic observation
Growth and Isolation
12-24h
Empirical antibiotic treatment
Patie
nt
If positive
Bloo
d sa
mpl
e
(5 to
10m
L)
Appropriate
dilution
Possible treatment modifications
Low contamination
(1 CFU / 10mL)
1. Assessment of bacterial presence
2. Bacteria isolation on solid media
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Current procedure for microbial identification
5
Intr
oduc
tion
A delay or a misuse in antibiotic treatment (in case of antibiotic resistant bacteria) results in an augmentation of patient deaths6,7.
6Davey, P.G. et al., Clinical Microbiology and Infection, 2008 ; 7Kumar, A. et al., Chest, 2009.
Hemoculture
12-36h
Gram coloration and
Microscopic observation
Growth and Isolation
12-24h
Identification and
Antimicrobial Susceptibility Testing
24h - 72h
Empirical antibiotic treatment72h – 96h
Suitable antibiotic treatment
Treatment adjusting
Result
Patie
nt
If positive
Bloo
d sa
mpl
e
(5 to
10m
L)
Appropriate
dilution
Possible treatment modifications
Low contamination
(1 CFU / 10mL)
1. Assessment of bacterial presence
2. Bacteria isolation on solid media
3. Full identification of the causative bacteria
Imperative need to shorten diagnosis time
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Goal : to perform hemoculture and identification in the same timeIn
trod
uctio
n
Growth and Isolation
12-24h
Identification confirmation and
Antimicrobial Susceptibility Testing
24h - 72h
Empirical antibiotic treatment72h – 96h
Suitable antibiotic treatment
Treatment adjusting
Result
Patie
nt
If positive
Bloo
d sa
mpl
e
(5 to
10m
L)
Appropriate
dilution
Possible treatment modifications based
on identification results
Low contamination
(1 CFU / 10mL)
1. Hemoculture
AND
identification2. Bacteria isolation on solid
media3. Full identification of the causative
bacteria
Hemoculture
AND
Identification
6
To obtain reliable identification results during hemoculture
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Use of protein biochip and optical detection
7
Intr
oduc
tion
Grafting of bacteria specific antibodies by a simple electrochemical reaction.
Antibody array
Cuve
Glass Prism
Gold layer
Samples 1 & 2
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Use of protein biochip and optical detection
8
Intr
oduc
tion
Grafting of bacteria specific antibodies by a simple electrochemical reaction.
Antibody array
Cuve
Glass Prism
Gold layer
Antibody grafted to the surface
ReactorWet
phasePrism
Dry phase
Assets of the SPRi:
•Direct and multiplex detection
•Label-free
•Real time monitoring
Samples 1 &2
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Culture – Capture – Measure approach8,9 by SPRi
9
Intr
oduc
tion
Detection in simple and complex media (food matrix)
Non destructive method which enables further testing (plating, PCR…) after incubation time.
Blood dilution with suitable culture media and artificial contamination
8Bouguelia, S. et al., Lab on a Chip, 2013 ; 9Mondani, L et al., JAM, 2014.
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Proof of concept in blood
10
Expe
rimen
tal r
esul
ts
Detection of 100 UFC.mL-1 of Salmonella enterica serotype Enteritidis in diluted human blood (mean of 3 spots)
Salmonella detection in blood is feasible in a few hours.
What happens with S. aureus?
Specific signal
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S .aureus detection in culture media
11
Expe
rimen
tal r
esul
ts
IgG control (non specific of S. aureus) looks positive.
Simultaneous interaction on all antibodies
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S .aureus detection in culture media
12
Expe
rimen
tal r
esul
ts
IgG control (non specific of S. aureus) looks positive.
Difficult to say if an antibody recognizes its target or if interactions are only "protein A" related.
Cause : Staphyloccocal protein A recognizing the Fc fragment of antibodies.
Simultaneous interaction on all antibodies
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IgG cleavage for S. aureus detection
13
Expe
rimen
tal r
esul
ts
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IgG cleavage for S. aureus detection
14
Expe
rimen
tal r
esul
ts
Anti-S. aureus digested IgG are successfully binding to their target
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IgG cleavage for S. aureus detection
15
Expe
rimen
tal r
esul
ts Workable but enzymatic digestion must be adapted to each antibody.
Anti-S. aureus digested IgG are successfully binding to their targetNon specific digested IgG are no longer recognized.
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Conclusion
Specific antibodies are required for proper bacterial recognition
Influence sensitivity and specificity of the assay.
Bacteria detection on an antibody array by SPRi is working in a few hours.
Easy to operate and applicable in complex media (diluted blood sample)
16
Conc
lusi
ons
& P
ersp
ectiv
es
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Perspectives
Mammalian antibodies could be (partially) replaced by alternative probes such as:
•Chicken antibodies
•Aptamers
Work to be done : •Screening of specific probes
•Analytical comparison with existing devices
17
Conc
lusi
ons
& P
ersp
ectiv
es
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ThanksCREAB : My PhD supervisors, Yoann Roupioz (PhD) and Thierry Livache (PhD).D. Pulido for the work done together. CHU Grenoble : Pr M. Maurin and S. Boisset (PhD)
The CEA programme « Technologies pour la santé » for the funding of my PhD thesis.
18
Thank you for your attentionAny questions?
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Microorganisms responsible of BSI
19
Anne
xs
From 5Timsit, J.F., et al., BMC Infect Dis, 2014, 14,
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Experimental device
20
Device placed in an incubator with temperature fixed at 37°C
Experiment monitored on a dedicated software
Prism and cuve with 2 chambers.
Optical bench of the SPRi system.
Anne
xs
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SPRi Principle
21
Anne
xs
Antibody grafted to the surface
LED CCD camera
Bacteria
Computer
Incident light
Reflected light
Resonance angle Θ
Wave penetration at the interface
Plasmon surface wave
Polarizer
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SPRi Principle
22
Anne
xs
Resonance angle shift with interaction
ΔR
(%)
Incident Light Angle (°)
Refle
ctivi
ty (%
)1. Resonance angle Θ2. Fixed working angle
1 2
Initial plasmon curvePlasmon curve after interaction