direct organism identification from positive blood ... harris.pdf · proteus mirabilis 4 4/4 4/4...

Direct Organism Identification

From Positive Blood Cultures

using MALDI-TOF Biotyper

(Bruker Daltonics GmbH)

Rhian Harris

Contents

• What is MALDI-TOF and how does it work?

• Aims

• Introduction

• Methods

• Results

• Discussion and conclusions

• Future developments

What is MALDI-TOF and how does

it work?

• MALDI - TOF is an

abbreviation for Matrix assisted

laser desorption/ionisation -

time of flight

• The instrument consists of a

platform, a tube, a laser and a

detector.


it work?

• The organism which is being identified smeared onto the steel plate

• Fixed with matrix

• Cells are ionised using a laser which is fired at the plate

• Laser fires 240 times


it work?

• The ions present are then accelerated up the tube

• The greater the molecular weight of the ions the longer the time of flight

• The time it takes for the ions to reach the detector is measured and converted to Daltons(Da)/molecular weight.

• Each ion will represent a peak on the graph


it work?


it work?

• Collectively will form a unique fingerprint for each organism

• The spectra produced is compared to the Bruker database of approximately 4111 organisms with different spectra and scored

• An identification is given in approximately 30-60 seconds

Scoring system

Three Calculations

Unknowns in reference (6/10)

Reference peaks in unknown

(6/20)

Score of relative intensities of

matching peaks (out of 10)

6 x 3 x Intensity Score

Total score out of 1000

Log10 score out of 3

Scoring

Scoring system

New Scoring system for Blood Cultures

Old Scoring New Scoring Meaning

>2.0 >1.8 Highly Probable Species

Identification

1.7-2.0 1.6-1.8 Probable Genus Identification

<1.7 <1.6 Non Reliable Genus

Identification

Aim

• The aim of this study was to determine if the Bruker

Sepsityper kit was able to successfully identify the micro-

organisms present in positive blood cultures by direct

extraction using Bruker Biotyper MALDI-TOF technology.

• The potential value of earlier species identification with

regard to clinical management and potential escalation

or de-escalation of antimicrobial treatment.

Introduction

• Blood cultures are an essential laboratory diagnosis tool to diagnose and determine treatment of a potential bacteraemia.

• The current method of detection of a positive blood culture involves the use of BioMerieux BacTalert system.

• Once removed a Gram film is carried out to determine the presence of any potential pathogens, and cultures performed to confirm the identification of the pathogen by BD Phoenix, a process which can take up to 48 hours.

• Early species identification may have clinical management implications and may also affect the antimicrobial therapy administered to the patient.

• MALDI-TOF mass spectrometry can revolutionise this process.

• MALDI-TOF-MS applied to positive blood cultures can yield a species identification result in <1 hour.

Introduction

• 78 Blood cultures

• 55 cultured organisms

• Processed using the Sepsityper kit

• Partly processed at RGH and Royal

Liverpool

• Processed every positive BC

Current method of identification

used in the lab

MALDI Sepsityper Method

Sepsityper BC method

• 1ml of Blood fluid

• 200µl of Lysis buffer

• Centrifuge for 1 min at 13,000 rpm

• Remove supernatant

• Re-suspend pellet with 1ml of washing buffer



• 300µl deionised water and 900µl ethanol

• Samples were then frozen and taken to Liverpool for processing

Sepsityper BC method




• Dry ethanol pellet

• Between 2 - 50µl formic acid added depending on size of pellet, and an equal volume of acetonitrile


• 1µl of the supernatant is spotted onto the target plate

• When dried the matrix was then added

ResultsOrganism No No ID by Maldi No ID by PHX Therapy Therapy Altered

E.coli 24 23/24 24/24 23/24 0/24

Staph.epidermidis 35 33/35 23/23 8/35 2/35

Candida albicans 2 1/2 0/2 0/2 0/2

Proteus mirabilis 4 4/4 4/4 4/4 0/4

Staph.pettenkeri 2 2/2 Failed to grow on culture 2/2 0/2

Strep.sanguinis 3 3/3 3/3 3/3 0/3

Strep.pneumoniae 5 4/5 0/5 No data No data

Strep.cristatus 2 2/2 0/2 1/2 0/2

Bacteroides vulgaris 2 1/2 0/2 No data No data

Klebsiella pneumoniae 3 3/3 3/3 3/3 0/3

Staphylococcus hominis 8 8/8Not identified as clinically

insignificant 4/8 0/4

Coryne.pseudodiptheriticum 2 2/2 1/2 0/2 0/2

Propionobacterium acnes 1 1/1 Failed to grow on culture 0/1 0/1

Klebsiella oxytoca 8 8/8 8/8 No data No data

Staphylococcus aureus 13 13/13 13/13 8/13 0/8

Enterococcus faecalis 2 2/2 2/2 2/2 2/2

Streptococcus salivarus 2 2/2 0/2 No data No data

Corynebacterium .acolens 2 2/2 0/2 0/2

Staphylococcus haemolyticus 2 2/2Not identified as clinically

insignificant 2/2 0/2

Micrococcus luteus 2 2/2 2/2 0/2 0/2

Nocardia farcinia 2 0/2 0/2 No data No data

Bacteroides sp 1 0/1 0/1 No data No data

Corynebacterium species 2 0/2 2/2 0/2 0/2

Misc Gram bacilli 4 0/4 0/4 No data No data

Total 133*

Results

Blood cultures new scoring system

79.49%

6.41%

14.10%

Direct Culture new scoring system

92.86%

7.14%

MALDI successfully identified 86.0% of organisms direct from blood

culture and 92.9% of organisms by extraction

Results

• Identified organisms present in the blood culture that

failed to grow

• Possible explanantions why some organisms did not

have identification

- Charcoal bottle

- Scanty organism present

- Organism not present in the database

- Mixed culture

Discussion

• MALDI-TOF successfully identified 85.9% of isolates direct from blood culture bottles in less than one hour.

• As demonstrated in this project the extraction of the organism direct from the blood culture can be taken to the storage of ethanol stage and then stored for processing at a different laboratory if necessary. This means that hospital sites without a MALDI-TOF can still use the technology.

• MALDI-TOF is very cost effective compared to our current identification system. Direct blood culture analysis costs £2-£4 per isolate and direct colony smear only 5p. Our current method costs £5 per isolate and cannot be done using positive blood direct from the bottle so result is only available on day 2.

• The new scoring system developed by Bruker allows greater confidence with the identification obtained by MALDI-TOF MS and as our results show it allowed us to confidently identify 11.54% more isolates.

Impact on Clinical Management

• 2 cases showed a potential to change antibiotic therapy due to results being available at an early stage.

• In one case Enterococcus faecalis was not identified until day 2 by our current method. Having a day 1 result would have resulted in a change of therapy.

• In a second case antibiotic therapy was given on the basis of GPC in the Gram film. The organism was identified to be S.epidermidis. Day 1 identification would have led to a de-escalation of antibiotic therapy as the organism was not significant in this case.

• Based on available clinical data the majority of cases to date in this study would not have yielded an escalation or de-escalation of antibiotic therapy however from a specialist point of view antibiotic management/stewardship will be aided by -– a) Day 1 identification of S.aureus to distinguish it from Coagulase Negative Staphylococci to

result in more focussed early therapy as well as avoidance of unnecessary antibiotics.

– b) Identification of Pseudomonas, Serratia, Enterobacter spp and Citrobacter as first line empirical Gram Negative Therapy is not generally directed at these organisms.

Future developments

• Future developments could be to directly

extract organisms from other fluids e.g.

CSF and synovial fluid to obtain a direct

organism identification and diagnosis of

infection and TB.

• Implementation into routine laboratory

• Potential to carry out typing with new

software

Thank you

• Thank you to Bruker for sponsoring the MSc project

• I would like to thank Craig, Erika and Michael for all the help and their continued support whilst carrying out the MSc

• Thanks to the staff at the Royal Liverpool Hospital for allowing us to work there

• Thank you to Kelly Ward and Jen Hancock for their help and support

Thank you for listening any

questions??

direct organism identification from positive blood ... harris.pdf · proteus mirabilis 4 4/4 4/4...

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