Impact of MALDI‐TOF on AST

NATHAN READINGSENIOR BIOMEDICAL SCIENTIST

MICROBIOLOGYSANDWELL AND WEST BIRMINGHAM HOSPITALS NHS TRUST

NATHAN.READING@NHS.NET@NATHANR999

ANTIMICROBIAL SUSCEPTIBILITY TESTING METHOD USER DAY 8TH JULY 2014

Overview• Context• Susceptibility Testing – Implications Of MALDI‐TOF.

• MALDI‐TOF and ART – practicalities• Influencing Antimicrobial Prescribing• Faster Sensitivity Testing?• Conclusions. 

About us 

• Large District General Hospital Trust• c.900 beds (Split across two hospital sites )• Serves approx 400,000 patients• Tertiary referral Ophthalmology inc A&E• Unified microbiology lab on single site• Previous home of BSAC Standardized Susceptibility Testing Method Development Centre. • Key interest in A.S.T.

Why MALDI‐TOF at City Hospital?• Efficiency

• Redesign our workflow

• Reconfiguration of service providers locally• 7 other microbiology laboratories within 10 miles

• 4 within 5 miles!

• Improvement of our service for users.• Decrease time to detection of pathogens

• Improved decision support

• Standardise our ID methods to a single platform

• Opportunity to get first VITEK – MS in U.K. clinical laboratory.

Why VITEK‐MS MALDI‐TOF at City Hospital?• Current VITEK 2 User – AST and GN/GP ID• Easy integration to attach ID to sensitivity testing using MYLA middleware 

solution.

• MYLA also has connectivity to our automated sample processing and blood culture system• Middleware is now potentially a powerful management tool• Smart Incubators/Sample preparation in the future

• Very simple ID protocol• Minimal extraction (Formic Acid for Yeasts only – done on target)• Simple sample preparation on disposable targets

Why is MALDI‐TOF important for AST/ART?

• Accurate identification can be used to predict phenotype• Inducible AmpC production.

• Morganella, Serratia, Enterobacter, Citrobacter, Providencia

• Ampicillin Resistant, Co‐amoxyclav Resistant, 3G ‐Ceph

• Intrinsic Vancomycin Resistance• Some Enterococci (VanC)

• Colistin Resistance• Serratia sp

• Stenotrophomonas, Elizabethkingia, Chryseomonas• Carbapenem = R

Why is MALDI‐TOF important for AST/ART?• Most breakpoint tables, zone diameter and MIC built using SPECIFIC species.• BSAC

• EUCAST

• If the species isn’t named or part of a group then there is no specific guidance!

• Use of foot notes and species guidance• Aminoglycosides, Ciprofloxacin, Cefuroxime and Salmonella sp.

• Vancomycin for Coag Neg Staphs vs S.aureus

Validation. Do we trust the ID provided?

• CE/FDA ‐IVD  Only database – reduced need to validate?• Performance claims assured?

• Non conformity or non performance addressed via regulatory bodies ‐MHRA?

UKAS/CPA  Stds F1.1, F1.2  and ISO 15189

• Non CE‐IVD Database – Extensive and robust validation?• Implications when using with patients?

• Approximately 1400 ‘highly characterised’ strains

• 93.8% ‐ 100% Species ID correct

• 97.4% ‐100% Group/Family Level ID Correct

• 98.5% ‐100% Genus Level ID Correct

Reading et al. ECCMID 2012  Poster P2323

Reading et al. ECCMID 2012 Poster P2332

Symonds et al. ECCMID 2012 Abstract R2637

Symonds et al. Microbiology Poster Session, IBMS Congress 2013, Poster 40 

Validation

Validation ‐ Direct Comparison 

• 400 further isolates• Parallel testing with current

• API• VITEK 2• Phenotypic/Biochemical/Microscopy

• No misidentification when compared to traditional• Limitations of database

• Strains not included• Slash line ID?

Our ROUTINE workflow – time saving?

When was ID available compared to traditional method?

Same 400 isolates – ‘REAL WORLD’ performance

Time to Identification

Organism Traditional (mean/range)/hours

MALDI‐TOF(mean/range)/hours

Staphylococci 19/15‐29 1.5 / 0.3‐4GNB (Ferm/Non Ferm) 10 /6‐22   , 13/8‐36 1.5/ 0.3‐3 , 2/ 0.5‐4Streptococci 8/ 0.1‐32 1.5/ 0.3‐4HACEK 18 / 17‐39 1.3/ 0.3‐3Yeasts 34 / 18‐68 1/ 0.3‐5Enterococci 10 / 9‐27 1.3/ 0.3‐4

Our ROUTINE workflow

• MS is now ‘de‐facto’ first line ID• No latex for Staphylococci and Streptococcal Grouping

• No Dnase plate

• No Germ Tubes

• API/VITEK 2 only on occasion, Shigella spp, S.typhi.

• ID set up and results reviewed by bench readers as cultures read.• Processed in groups of 16 isolates

• Organism ID ready for choice of sensitivity testing.

Our ROUTINE workflow – Lab Aspects• Decrease turnaround times

• Bacterial Identification available earlier.

• Standardised identification platform• Reduction of identification methods

• Reduction of training requirements

• Reduction of reagent requirements• Reduced transport & packaging ‐ sustainability

• Reduced storage space/refrigeration requirements

• Increased throughput capacity• Addresses pathology reconfiguration requirements

• Locally and Nationally

Efficient susceptibility testing• Effective Identification

• Only test appropriate isolates.• Reduction of wastage

• Non S.aureus strains but Staphylococcal Latex Positive, Dnase equivocal/positive

• Staphylococcus lugdunensis• Oxidase positive non‐Pseudomonas non‐fermenters

• Only test appropriate antibiotics• Specific panels based on Genus vs general broad panels

Our workflow savings/improvements• ?S. aureus

• Latex and DNase• 2‐3 Isolates per day through Vitek 2  NOT S. aureus

• Cost approximately £5.00 per isolate (Purity plate, Vitek  2 Consumables, Staff Time)

• 1 week study 11 Isolates non S. aureus

• Extrapolated saving £2800 p.a.• ?S. pneumoniae

• Optochin/Bile Solubility

• Approximately £1.15 per isolate (Plate, Discs, Staff Time)

• Approximately 20% not S.pneumoniae

• Extroplated saving £350 p.a.

Efficient susceptibility testing• Can choose most appropriate testing method

• Automated – Vitek 2•Staphylococci, Enterobacteriacae, Pseudomonads, Enterococci

• BSAC Standardized Disc Method•Streptococci (AHS, BHS, S.pneumoniae)•Neisseria meningitidis, N. gonorrhoeae•Moraxella catarrhalis, Haemophilus influenzae

• Gradient Strips / Referral •Everything else!

Workflow – capacity extension

• Prior to MALDI  April 2009‐ November 2011• VITEK 2 at 75%‐ 100%   Mon – Fri

• ID and Sensitivity Testing

• Ongoing workload increases

• Post MALDI November 2011 ‐ Present• Vitek 2 at 75% ‐100% Mon‐Fri

• Sensitivity Testing Only

• Second instrument not required

WORKFLOW EFFICIENCY ‐ STAFFING

• AST  Pre Vitek 2• 1 WTE Senior BMS, 2 WTE Band 6 BMS• 1 WTE Lab Support Work

• AST Post Vitek 2 Pre MALDI• 1 WTE Senior BMS, 1 WTE Band 6 BMS• 0.5 WTE Lab Support Worker

• AST Post Vitek 2 Post MALDI• 1 WTE Band 6 BMS• 0.5 WTE Lab Support Worker

• Accurate ID – better characterisation of infecting species• Increased likelihood of reported isolate

• Needs susceptibility testing

• How?• Discs x  EUCAST/BSAC  more for CLSI but zone diameter cut‐offs limited

• Vitek/Phoenix x

• Sensititre/Microscan

• ISO Microbroth?  Yes but

• No interpretation

• Off line read?

Problems? Testing without guidelines/methodology?

Problems? Testing without guidelines/methodology?

Agar/Broth MIC ‐ Yes • Need antibiotics• Adequate controls• Time consuming• Specialist equipment/Reference

Gradient Strips ‐ Yes•Care with some antibiotics•Cost and Range

Problems? Testing without guidelines/methodology?

• How to interpret?• Breakpoint tables

• BSAC/EUCAST Anaerobe tables

• EUCAST species non‐specific table• ECOFF’s – via EUCAST website

• Look at MIC distributions• Look to Case Reports

How do we speed up advice for Sepsis?

• Sample cultured – 24 hours – ID Set up ‐ 24 hours• 48 Hours to correct antibiotic guidance

• Sample cultured ‐24 hours –Identification Available• 24 hours to correct antibiotic guidance

• Blood culture sampled – 24hours – Flags Positive• Gram Stain 24 hours.

• Subculture ‐48 hrs ID

Extending the use of MALDI TOF.

• ECCMID 2011, Milan• Poster P1825• 97.8% Spiked Bottles

• Fothergill A. et.al.• J.Clin.Microbiol. March 2013;51:3 805‐809

• 72.6% Clinical Samples

Reading et.al. ECCMID 2013 Berlin, E‐Poster eP769

Impact of Direct Blood Culture ID using MALDI TOF.

• MALDI‐TOF direct identification, would have led to modification of empiricaltherapy of 18 patients (12.32%)

• If TNase media not used then potential for a further 47 patients to have beenpositively impacted. Total potential impact 67/146 patients (44.5%)

• All 16 bottles of AmpC‐producing Enterobacteriaceae species from 8 patientswere correctly identified at the species level by MALDI‐TOF.

• Direct identification for Enterobacter spp. would have lead to broadening ofantibiotic in 6 patients. In 5 of these patients, there was delay in startingappropriate antibiotics for 48 hrs.

• One patient with Enterobacter spp. bacteraemia died 24 hrs. after startinginappropriate antibiotic for sepsis.

• Identification on day 1 likely to have had a definite impacton patient management.

Impact of Direct Blood Culture ID using MALDI TOF.

• Impact of Matrix‐Assisted Laser Desorption Ionization Time‐of‐Flight Mass Spectrometry on the Clinical Management of Patients With Gram‐negative Bacteremia: A Prospective Observational Study

• Clerc. O et al. Clin Infect Dis. (2013) 56. (8):1101‐1107

MALDI‐TOF identification led to a modification of empirical therapy in 71 of all 202 cases (35.1%), and in 16 of 27 cases (59.3%) of monomicrobial bacteremia caused by AmpC‐producing Enterobacteriaceae.

Extending the use of MALDI in AST.• Identification of organism in positive blood cultures

• Wider range of organisms vs. FISH Microscopy and CE Marked Multiplexed Molecular SEPSIS Assays

• Cheaper• Very quick – 30mins!• Same day effective clinical advice• Tailored therapy – knowledge based vs. best guess.

• Better patient outcomes?• Contaminant vs. Pathogen – De‐escalate therapy earlier

• Pharmacy savings?• Tailored direct sensitivity testing vs. wider range of panels

• Reduces cost.

Resistance testing  6 ‐18 hours

Faster sensitivity testing

• Molecular• Limited Targets

• Target deviation/Novel variants

• Genotype doesn’t always predict Phenotype• EUCAST Expert rules for Cephs/Carbs

• If it tests S then in theory can treat…….?????

• Expensive• Skilled workflow

• GeneXpert, BD‐Max

• Digital Microscopy

• Micro‐fluidics• Sensitivity testing reported down to 1‐2 hours

• ‘Early’ Disc reading• 6‐8 hours post inoculation

• Needs re‐evaluated zone diameters

• EUCAST Eurostar Rapid Disk Method

• ECCMID 2013 Sundqvist M et al, P1541

• Automated zone reading built into Smart Incubators• BD Kiestra/ Copan/ Biomerieux

• Driven by smart image software with expert rules?

• Read when ready?

Faster sensitivity testing

Rapid phenotypic tests?

HMRZ ‐86BioRad Beta LactaMast Cica Beta

Carba NP TestRosco Rapid Carb ScreenRapidec Carba NP

MALDI-TOF - Detection of Metabolites produced after enzyme hydrolysis

+ Enzyme

Antibiotic Enzyme hydrolysis

E

Studies: • B‐lactam antibiotics 

• Ampicillin, Ertapenem, Meropenem, 3‐G Cephs

Antibiotic resistance screening.

MBT_MSBL assay

33

+ 18 Da - 44 Da

05 August 2014

• Functional assay, detection of hydrolysis of betalactam antibiotics

• Rapid: 1-4 hours

• Simple pipetting protocol

• Same instrument like for identification

• A number of publications show performance

The first useful MALDI-TOF MSresistance test

→ get it into rou ne!

MALDI Biotyper

Targetpreparation

Bacterial suspensionin antibiotic

solution

Incubation1 -4 h, 37°C

centrifugationsupernatant

Data interpretation

MBT_MSBL assay

3405 August 2014

05/08/2014 35

Ampicillin hydrolysis

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MBT_MSBL Publications

Hrabák J, Walková R, Studentová V, Chudácková E, Bergerová T. (2011) Carbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 49: 3222-7

Burckhardt I, Zimmermann S. (2011) Using matrix-assisted laser desorption ionization-time of flight mass spectrometry to detect carbapenem resistance within 1 to 2.5 hours. J Clin Microbiol. 49:3321-4.

Sparbier K, Schubert S, Weller U, Boogen C, Kostrzewa M. (2012) MALDI-TOF MS based functional assay for the rapid detection of resistance against ss-lactam antibiotics. J Clin Microbiol. 50:927-37

Hrabák J, Studentová V, Walková R, Zemlicková H, Jakubu V, Chudácková E, Gniadkowski M, Pfeifer Y, Perry JD, Wilkinson K, Bergerová T. (2012) Detection of NDM-1, VIM-1, KPC, OXA-48, and OXA-162 carbapenemases by MALDI-TOF mass spectrometry. J Clin Microbiol. [Epub ahead of print]

Kempf M, Bakour S, Flaudrops C, Berrazeg M, Brunel JM, Drissi M, Mesli E, Touati A, Rolain JM.(2012). Rapid detection of carbapenem resistance in Acinetobacter baumannii using matrix-assisted laser desorption ionization-time of flight mass spectrometry. PLoS One 7:e31676

Álvarez-Buylla A, Picazo JJ, Culebras E. (2013) Optimized method for Acinetobacter species carbapenemase detection and identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 51(5):1589-92

Wang L, Han C, Sui W, Wang M, Lu X. (2013) MALDI-TOF MS applied to indirect carbapenemase detection: a validated procedure to clearly distinguish between carbapenemase-positive and carbapenemase-negative bacterial strains. Anal Bioanal Chem. 405(15):5259-66

Lee W, Chung HS, Lee Y, Yong D, Jeong SH, Lee K, Chong Y. (2013) Comparison of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry assay with conventional methods for detection of IMP-6, VIM-2, NDM-1, SIM-1, KPC-1, OXA-23, and OXA-51 carbapenemase-producing Acinetobacter spp., Pseudomonas aeruginosa, and Klebsiella pneumoniae. Diagn Microbiol Infect Dis. [Epub ahead of print]

05 August 2014

05 August 2014 37

Web-based MSBL software prototypeSTAR_BL

38

Workflow of the MBT_MSBL assayfrom positive blood cultures

MALDI Biotyper

Targetpreparation

Positiveblood

culture

1 ml culture

Resuspensionin antibiotic

solution

Incubation1 -2 h, 37°C

centrifugationsupernatant

ModifiedSepsityperprotocol

Data analysis

05 August 2014

MALDI–TOF Mass Spectrometry for Rapid Antibiotic Resistance Detection - Mirande,C et al

Carbapenemase

Inactive Faropenem (hydrolysed) Carbapenem-S Peaks @ 308.3 & 330.3 m/z

Carbapenem-R Decreased intensity of peaks @ 308.3 & 330.3 m/z Additional peaks @ 304.3, 326.3 & 348.3 m/z

Faropenem Klebsiella pneumoniae 3hrs incubation @ 37oC + Faropenem Centrifuge 2 min 13,000g

Carbapenem-S strains (Neg controls)

Carbapenem-R strains(Carbapenemase producers)

E

MBT MS_RESIST

40

patent allowed & patent pending

05 August 2014

Resistance testing with stable isotopes

Growing cell perform protein biosynthesis

Integration of labeled amino acids resulting in peaks shifts

Monitoring growth in the presence of antibiotics by analyzing peak shifts

normal Lys Control 1

heavy Lys Control 2

heavy Lysantibiotic

Resistant

Susceptible

Principle of the MS_RESIST

4105 August 2014

MBT MS_RESIST

42

S. aureus strains, mass spectra – gel view

05 August 2014

A: Susceptible strain B: resistant strain„normal“

„heavy“

„heavy + Oxa“

„normal“

„heavy“

„heavy + Oxa“

WHAT ELSE?• Typing 

• Genus specific

• Some evidence of clustering of similar strains

• Not currently routine

• Many of the studies on small numbers

• Specific resistance markers PBP2’, Van A/B• Current systems in routine not currently

• Some work on MRSA very early on

• Confounding studies

• Carbapenem Resistance in Bateroides fragilis• Nagy et al.

• Delineate strains Carbapenem resistant from sensitive CfiA positive vs CfiA negative

Antibiotic resistance screening?• Sufficiently robust and accurate for ROUTINE?

• Good performance with known isolates and in ‘expert’ labs

• Maybe good, cost effective option in outbreaks• Delineate ESBL/AMPC + Impermeability vs

Carbapenemases• Less active enzymes?

• Hydrolysis can be slow• Labile substrates• Experience of Carbapenemases and AST?• Yes/No simplified result.• Advantages over molecular confirmation?

• Striving for robust ROUTINE identification• Good quality databases which are CE‐IVD marked

• Enables trust in identification, secure clinical decisions

• Need speed of Resistance Testing to catch up• New technologies

• Re‐evaluation of existing?

• Ensuring Resistance Testing is performed accurately in context to identification

• Right method for right organisms

Conclusions

THANK YOU!

• Nathan.Reading@nhs.net• @nathanr999