Download Towards rapid detection of Staphylococcus aureus during blood

Towards rapid detection of
Staphylococcus aureus during blood culture
Vincent Templier, PhD Student
CEA, INAC-SPrAM-CREAB, F-38000 Grenoble, France
Contact : [email protected]
World Congress and Expo on Applied Microbiology
August 18-20, 2015, Frankfurt
The threat of bacteremia
• Bacteremia or bloodstream infection (BSI) = presence of viable bacteria in
blood1,2.
• Affects mainly immunocompromized patients but not only.
Introduction
• 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
1. Assessment of
bacterial presence
(5 to 10mL)
dilution
Hemoculture
12-36h
Low contamination
(1 CFU / 10mL)
Patient
Introduction
Blood sample
Appropriate
Empirical antibiotic treatment
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Current procedure for microbial identification
1. Assessment of
bacterial presence
2. Bacteria isolation on solid
media
(5 to 10mL)
dilution
Low contamination
(1 CFU / 10mL)
Hemoculture
If positive
Growth and Isolation
12-36h
12-24h
Gram coloration
and
Microscopic observation
Possible treatment
modifications
Patient
Introduction
Blood sample
Appropriate
Empirical antibiotic treatment
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Current procedure for microbial identification
1. Assessment of
bacterial presence
2. Bacteria isolation on solid
media
(5 to 10mL)
dilution
Low contamination
(1 CFU / 10mL)
Hemoculture
If positive
Identification and
Growth and Isolation
12-36h
12-24h
24h - 72h
Gram coloration
and
Microscopic observation
Possible treatment
modifications
Patient
Antimicrobial Susceptibility
Testing
Empirical antibiotic treatment
Result
Introduction
Blood sample
Appropriate
3. Full identification of the causative
bacteria
Treatment
adjusting
72h – 96h
Suitable antibiotic
treatment
A delay or a misuse in antibiotic treatment (in case of antibiotic resistant
bacteria) results in an augmentation of patient deaths6,7.
 Imperative need to shorten diagnosis time
6Davey,
P.G. et al., Clinical Microbiology and Infection, 2008 ; 7Kumar, A. et al., Chest, 2009.
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Goal : to perform hemoculture and identification in the same time
1. Hemoculture
AND
identification
2. Bacteria isolation on solid
media
3. Full identification of the causative
bacteria
(5 to 10mL)
Blood sample
dilution
Hemoculture
AND
If positive
Growth and Isolation
12-24h
Identification
Identification confirmation
and
Antimicrobial Susceptibility
Testing
24h - 72h
Low contamination
Patient
(1 CFU / 10mL)
Possible treatment
modifications based
on identification
results
Empirical antibiotic treatment
Result
Introduction
Appropriate
Treatment
adjusting
72h – 96h
Suitable antibiotic
treatment
 To obtain reliable identification results during hemoculture
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Use of protein biochip and optical detection
Samples 1 & 2
Gold layer
Glass
Prism
Antibody array
Introduction
Cuve
Grafting of bacteria specific antibodies by a simple electrochemical reaction.
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Use of protein biochip and optical detection
Samples 1 &2
Gold layer
Glass
Prism
Antibody array
Introduction
Cuve
Grafting of bacteria specific antibodies by a simple electrochemical reaction.
Antibody grafted
to the surface
Reactor
Wet
phase
Prism
Dry phase
Assets of the SPRi:
• Direct and multiplex detection
• Label-free
• Real time monitoring
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Culture – Capture – Measure approach8,9 by SPRi
Introduction
Blood dilution with suitable culture media and artificial contamination
 Detection in simple and complex media (food matrix)
 Non destructive method which enables further testing (plating, PCR…) after
incubation time.
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8Bouguelia,
S. et al., Lab on a Chip, 2013 ; 9Mondani, L et al., JAM, 2014.
Proof of concept in blood
Experimental results
Detection of 100 UFC.mL-1 of Salmonella enterica serotype Enteritidis in diluted
human blood (mean of 3 spots)
Specific signal
 Salmonella detection in blood is feasible in a few hours.
 What happens with S. aureus?
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S .aureus detection in culture media
Experimental results
IgG control (non specific of
S. aureus) looks positive.
Simultaneous
interaction on
all antibodies
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S .aureus detection in culture media
Experimental results
IgG control (non specific of
S. aureus) looks positive.
Simultaneous
interaction on
all antibodies
Cause : Staphyloccocal protein A recognizing the Fc
fragment of antibodies.
 Difficult to say if an antibody recognizes its target or if interactions are only
"protein A" related.
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Experimental results
IgG cleavage for S. aureus detection
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Experimental results
IgG cleavage for S. aureus detection
Anti-S. aureus digested
IgG are successfully
binding to their target
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Experimental results
IgG cleavage for S. aureus detection
Workable but enzymatic digestion
must be adapted to each antibody.
Anti-S. aureus digested
IgG are successfully
binding to their target
Non specific digested
IgG are no longer
recognized.
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Conclusion
Conclusions & Perspectives
 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)
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Perspectives
Conclusions & 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
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Thanks
CREAB : 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.
Thank you for your attention
Any questions?
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Annexs
Microorganisms responsible of BSI
From 5Timsit, J.F., et al., BMC Infect Dis, 2014, 14,
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Experimental device
Annexs
Prism and cuve with 2
chambers.
Optical bench of the SPRi system.
Experiment
monitored on a
dedicated
software
Device placed in
an incubator with
temperature
fixed at 37°C
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SPRi Principle
Bacteria
Antibody grafted
to the surface
Incident
light
LED
Reflected
light
CCD camera
Annexs
Computer
Plasmon surface wave
Wave penetration
at the interface
Polarizer
Resonance angle Θ
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SPRi Principle
1. Resonance angle Θ
2. Fixed working angle
Initial plasmon curve
Plasmon curve after interaction
ΔR (%)
Reflectivity (%)
Annexs
1 2
Resonance angle shift
with interaction
Incident Light Angle (°)
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