Mark Wilks, Clinical Scientist Barts Health Trust, London UK

MALDI-TOF in Diagnostic microbiology Mark Wilks, Clinical Scientist Barts Health Trust, London UK Barts and the London NHS Trust St Barts Hosp 112...
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MALDI-TOF in Diagnostic microbiology

Mark Wilks, Clinical Scientist Barts Health Trust, London UK

Barts and the London NHS Trust

St Barts Hosp 1123

(Royal) London Hospital 1740

Beds 1,200 Total number of attendances 960K Outpatient attendances 590K A&E patients 195K X-Rays and scans carried out 378K Kidneys y transplanted p 134 Pathology tests 7.9 million

London Chest 1860s

„

Merger with Whipp’s Cross and Newham NHS Trusts Æ Barts Health Trust April 2012Æ largest g UK Trust

Diagnostic Micro belongs to the late 19C! Robert Koch lab necessary to isolate suspected bacterial pathogens • cultured bacteria on the sterile surfaces of cut, boiled potatoes •regular liquid medium solidified by adding gelatin melted @ T>28°C •Fannie Eilshemius Hesse (1881) suggested use of agar; 100°C to melt, 40-50 °C to solidify. p p petri •Richard Petri ((1887)) developed dish, a container for solid culture media. •Gram stain 1882

MALDI-TOF: MALDI TOF: a revolution in microbial ID? -

MALDI: Matrix Assisted Laser Desorption Ionisation 377nm N2 Laser

Sample plate

1. Colony (C) is mixed with excess matrix (M) and dried on a MALDI plate. plate



CH+

2. Laser flash ionises matrix molecules. 3. Sample molecules are ionized by proton transfer from matrix: MH+ + C Æ M + CH+.

+20 kV

Variable Ground Grid Grid

Time-of-flight g mass analyzer y Source

Drift region (flight tube)

+

+

+

V

•Ions are formed in pulses. •Small ions reach the detector before large ones. •Measures the time for ions to reach the detector.

detecctor

+

Tanaka: Nobel prize acceptance speech: p

1996 two key papers showing use of MALDI for Bacterial ID , note use of variable low MW ranges, g , 2KDa available

Claydon:

Holland et al

Low influence of culture conditions Pseudomonas oleovorans grown on different media Psdm. oleovorans B396_Medium 360

1000 0

Psdm. oleovorans B396_Medium 464

1000 0

Psdm. oleovorans B396_Medium 53

1000 0

Psdm. oleovorans B396_Medium 65

1000 0

Psdm oleovorans B396 Psdm. B396_Medium Medium 98

1000 500 0

Psdm. oleovorans B396_MRS10

2000 1000 0

Psdm. oleovorans B396_YPD

2000 1000 0

4000

5000

6000

7000

8000

9000

10000

11000

m/z

4364.06

E.coli 6254 4.64

538 80.64

5000

7157.65 727 73.87

3000

6410 0.90

5096.01

6315.49

4000

78 870.62

2000

ribosomal Protein RL36 RS32 RL34 RL33meth. RL32 RL30 RL35 RL29 RL31 RS21

1000

8368.99

Intens. [[a.u.]

Why does it work? - differences in ribosomal proteins

0 4000

4500

5000

5500

6000

6500

7000

7500

8000

m/z

m/z 4364,33 5095,82 5380,39 6255,39 6315,19 6410,60 7157,74 7273 45 7273,45 7871,06 8368,76

MALDI new standard for bacterial ID • 90-95% of bacteria and fungi rapidly identified within minutes? cost effective • Improved Filamentous fungi methods • Improved anaerobes data base • Improved Mycobacterial ID • Identification of mixed cultures ? common reason for ‘no reliable ID’ - new algorithims may help • Detection from positive blood culture: good • Detection of antibiotic resistance: ? possible

Other markers: • -

- RNA chaperones cold shock proteins cell division proteins DNA binding proteins

Available MALDI-TOF MALDI TOF systems: •

• • • • •

Saramis Spectral Archiving and Microbial Identification System/ Anagnostec – BioM RUO super spectra sum of peak weights compared to mass signals for each super spectra --> confidence value two different matrices Biomerieux Vitek MS IVD CE - Advanced Spectrum classifier - supervised learning of 25K spectra - artificial neural network analysis

• • • • •

Bruker B k MALDI Bi Biotyper t IVD CE - multiple lti l spectra t ffrom a single i l strain t i 1. peaks in sample mass spectra compared to peaks in ref spectra 2. reverse peaks in ref spectra compared to peaks in sample mass 3 spectra signal intensies of matched spectra 3. scores multiplied --> normalised to a value of 1000 --> log 3.0



A d Andromadas d Nassif N if Univ U i off Paris P i Sud, S d runs on Bruker B k MS

• •



Try: Matrix -2,5-dihydroxy benzoic acid in acetonitrile: ethanol: water ((1:1:1)) with 0.3 % TFA ((DBA)) (AnagnosTec, May 2010) Fleximass

DS

MAB

Pictures from Stefan Bugovsky 22

Behaviour of Matrix α-CYANO on Target Plates (AnagnosTec, (AnagnosTec May 2010) Fleximass

DS

MAB

Pictures from Stefan Bugovsky 23

Martiny et al JCM Feb 2012

Costs ( CE IVD / RUO / FDA approval?) ?? Cost • Standalone: UK £115K Bruker +£12K pa, • BD Phoenix + Bruker ( Kiestra) • BioMerieux + Vitek AST •Need high throughput: Barts health: 150 ID per day, 1,000 per week, 50,000 pa • Barts Health ‘Saved’ 1 BMS post

Running costs? • Costs of steel plates/ disposable plates • Single Si l or d double bl spots t • HCCA M Matrix t i costs: t 5 -20p 20 per spott • Cheapest own brand matrix e.g. Sigma • Reconstitute, R i di divide, id kkeep iin d dark k iin tightly i hl stoppered tube • Bruker, Br ker reconstit reconstitute, te dissolves dissol es very er easily easil • BioMerieux, ready made up • Allow All ffor matrix t i wastage, t 10 10-20% 20%

Should I buy and which system part 2? • Assume successful ID of around 90% isolates • What about the remainder • What about pneumos/mitis/oralis differentiation • Vitek: yes Bruker: no (? yes in new software) • 16S does not help, wait for optochin/rapid bile test • Add another gene • Differentiation of E coli/Shigella not possible with Bruker or Vitek (? S Soon possible with Bruker)) • need to retain biochemical tests

16S ID of pneumos: p position 203 C in pneumo position 203 A in other mitis group

Errors: • Barts Health lab re-organisation – Reduced error rate in IDs, –p plate readingÆ g MALDI ÆMALDI result Æ p plate re-read by another worker – Plates are effectively double read – Guards against MALDI transcription errors

Æ AST • Calibrate each plate • Negative controls – matrix only • “How do you know it’s right?”

Chlamydia detection in Sweden: deletion in a cryptic ti plasmidÆ l idÆ ‘reduction’ ‘ d ti ’ in i chlamydia hl di

Chlamydia detection in Sweden:

? User friendly • Bruker: 3 ID methods – direct smear – Formic acid in situ lysis ( Haigh et al 2011) – Alcohol extraction – Stand St d alone l system t without ith t LIMS OK

• Vitek – Direct smear – formic acid for yeasts – Needs LIMS integration, then more rapid th Bruker than B k

Vitek: Full integration

LIS

VITEK®MS with VITEK®2 Workflow W kfl

Prep Station

VITEK® MS Prep Station

VITEK® 2 MS ID Database D t b MS-ID ( CE/IVD) embedded into Myla®

P ow er

Ionising

Va cuum

Neg ative

VITEK® MS

VITEK®MS with VITEK®2 workflow

VITEK® 2 Prep Station P ow e r

Io nisin g

V ac uum

N ega tiv e

2a.Slide a S de description

Myla® 3. Peak lists

Acquisition Station

LI S

Beware e aeo of e exaggerated agge ated cclaims a s • Generally not from manufacturers • Look for the ‘follow follow up paper” paper • eg differentiation of MRSA and MSSA? – Same sets used for training and then ID – Testing only local strains- MRSA clonal Therefore many strains in your area are v. similar

The problem of selecting correct databases • Saramis database RUO can add own spectra • VITEK IVD locked database

• Vitek separate p yyeast and bacterial databases • e.g. small white colony on blood plate ?CNS If yyou select the bacterial database Æ Lactococcus If you select the yeast database Æ Candida albicans



Bruker secure database •

Bruker separate ‘Secure database’ – B. anthracis, S typhi, Brucella, Y. pestis • e.g. GNB from a blood culture ? Brucella at another hospitalÆ Bruker MALDI Æ good spectrum but no ID • Did not have ‘secure database’ • Sent to Barts Health Lab run against secure databaseÆ Brucella • 1 month th llater, t same mistake i t k b by our llab b • did not select the right database Æ good spectrum but no ID Æ 16SÆ Brucella!! • Vitek: no Brucella

Speed ! – Combination C bi ti off llaser power – Time taken to acquire spectra • Fixed number of shots • Intelligent software can terminate acquistion early

• Barcode agar plates and scan in • Test for yourself - plate of 20 S aureus and 20 0 E co coli a and d ttime e run u • Bruker: plate of 96 E coli = 1h • Laser life span and cost of replacement • Distance between target plate and source • Shorter this distance the more cleaning needed!

Reliability e ab ty and a d support suppo t • Web based support pp essential • What is your back up plan for breakdown and service? • If you loose vacuum how long before usable? • Rely on neighbouring lab with same technology - swap plates • What are the engineers like – how many, g What were they y doing g what training? before MS!

Questions? • Email: [email protected] • PDFs of all papers will be available

Acknowledgements • Barts and the London Charity y for a g grant • • Staff St ff off the th Micro Mi lab l b att Barts B t Health H lth Conflicts of interest: Speaker honoraria, travel support: Bruker and BioMerieux Grant for costs of BC work from BioM Ad i Advisory b board d off Th Thermofisher fi h

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