The Implementation of MALDI-TOF for Clinical use in a District General Hospital
Kelly Ward Senior Biomedical Scientist Royal Glamorgan Hospital, Llantrisant, South Wales Contents
• Why MALDI-TOF? • MALDI-TOF Validation • Current Clinical Methodology • Implementation into routine work • Cost Savings? • Direct Blood Culture Analysis – Examples • Clinical Examples • Where are we now? • The Future Why MALDI-TOF?
First Publications..... Methods for Microorganism Identification based on MALDI-TOF-MS
Claydon MA, Davey SN, Edwards-Jones V, Gordon DB. The rapid identification of intact microorganisms using mass spectrometry. Nat. Biotechnol. 1996;14(11):1584-6.
Krishnamurthy T, Ross PL, Rajamani U. Detection of pathogenic and non-pathogenic bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 1996;10(8):883-8.
Holland RD, Wilkes JG, Rafii F, Sutherland JB, Persons CC, Voorhees KJ, Lay JO Jr. Rapid identification of intact whole bacteria based on spectral patterns using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 1996;10(10):1227-32.
Mass spectrometry is not a new technology! MALDI Biotyper References Global MBT Sites
Globally: 352 Europe: 300 US: 19 Canada: 2 Japan: 8 South Korea: 6 Singapore 2 Australia: 2 New Zealand 1 China: 5 Thailand: 2 India 2 Middle East 1 South Africa: 2
August 2011 United Kingdom and Ireland Biotyper Systems
MBT systems
August 2011- 23 Systems Recently the HPA decided to adopt MALDI and the following HPA regional network laboratories are using/waiting for their system – Cambridge Addenbrooke’s – Colindale – Birmingham Heartlands – Southampton MALDI Clinical Validation Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM, Raoult D (2009). Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis. 49:543-51. Eigner U, Holfelder M, Oberdorfer K, Betz-Wild U, Bertsch D, Fahr AM (2009). Performance of a matrix-assisted laser desorption ionization-time-of-flight mass spectrometry system for the identification of bacterial isolates in the clinical routine laboratory. Clin Lab. 55:289-96.
S. Q. van Veen, E. C. J. Claas, and Ed J. Kuijper (2010). High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) in routine medical microbiology laboratory. J. Clin. Microbiol 48:900-7
Cherkaoui A, Hibbs J, Emonet S, Tangomo M, Girard M, Francois P, Schrenzel J (2010). Comparison of two Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Methods with Conventional Phenotypic Identification for Routine Bacterial Speciation. J. Clin. Microbiol. 8:1169-75
Bizzini A, Durussel C, Bille J, Greub G, Prod'hom G (2010). Performance of Matrix-Assisted Laser Desorption/Ionization Time- of-Flight Mass Spectrometry for the identification of bacterial strains routinely isolated in a clinical microbiology laboratory. J Clin Microbiol. [Epub ahead of print]
MALDI Biotyper has ARRIVED in Clinical Routine!!!
In RGH – Had to Validate its suitability to fit into the workflow to maintain or increase the quality of our results Why MALDI-TOF?
• Primary- HEALTH BOARD HAS TO SAVE MONEY!!!
• Rhians B/C Project – demonstrated its advantages
• Phoenix – only combination panels available = COST (+ 50 panels/day)= £250.00/Day
• ID based on sugar reactions – standardised media needed for accurate results
• MALDI-TOF – after initial rental price – each sample tested = 5p
• Identification - measurement of high abundance ribosomal proteins/protein fragments – not influenced by environment or growth conditions
• Good clinical evidence available to demonstrate its accuracy in identifying clinical and environmental organisms. Methodology – Before MALDI
Cultured Bench ID after 24hrs Final ID – 48-+72hrs
URINE BENCH
B/C & MRSA BENCH •Colony Morphology •GRAM Film
PUS BENCH •Staphaurex Combination Panels •Strep Grouping Kit Only •Oxidase Reagent FAECES BENCH •Phadebact Kit •Anti-Sera RESPS BENCH •Etc, etc, etc
Referral to GENITAL BENCH Reference Lab if required MALDI-TOF Clinical Validation
We received our MALDI-TOF in August 2011 – FIRST LAB IN WALES TO USE MALDI CLINICALLY!! Three days training from Bruker and 4 staff chosen as ‘Trainers’ BEFORE INTRODUCTION TO BENCH WORK - Processed – 78 Gram Positive Orgs 114 Gram Negative Orgs 14 Yeast Variety of NTCC/ATCC Orgs •All results collaborated with the current methodology •System was easy to use •Results easy to interpret •SOP Written……………….Ready for Staff Training!! Staff Training
Staff were trained on a one to one basis and Training covered – -How MALDI-TOF worked! Staff understanding was important to ensure samples were correctly prepared and put through in the correct manner. -Demonstration & Practical experience of ‘Direct Smear Technique’
-Demonstration & Practical experience of ‘Matrix Addition’
-Demonstration & Practical experience of MALDI-TOF Biotyper Software & Wizard Staff Training Continued Completely new way of working! •Decision - use MALDI-TOF for organism identification & Disc Sensitivities (exception of B/C, Multi Resistant Organisms and Sterile Sites as full Phoenix sensitivity needed)
•Cwm Taf– Urine Bench is BUSIEST!! Prior to merge 150-200 samples/day. Post Merge – approx 300 samples/day
•View – to implement onto urine bench 1st – if it worked here it would work everywhere else
•Worked on urine bench to find best way to read plates, implementing MALDI-TOF & ensuring work completed in a timely manner - Benchmark written •LEAN Optimum– Only 24-36 samples put through on each run to prevent bottle neck at the instrument.
Fingers Crossed!!! MALDI-TOF Roll-out
OctOct NovNov Dec Jan Etc
Oct Staff 1 - Urines Staff 1 - Resps Staff 1 -Faeces Staff 1 -PUS
Training 2 days
Nov Staff 2 - Urines Staff 2 - Resps Staff 2 -Faeces
Training 2 days
Dec Staff 3 - Urines Staff 3 - Resps
Training 2 days
• Staff work at varying speeds. Question? Can everyone adopt it? – Answer - Staff 1 & 2 opposite ends of spectrum so proved it would work for everyone. •MALDI-TOF skills are transferable – No need for retraining on each bench as same principle applied. Disc Sensitivity applied to all ‘normal’ organisms. •Result – in 6 months there should be a staff member who is using MALDI-TOF in all laboratory areas. MALDI Numbers Tested to Date
Month Number Number Mean analyte measured measured number per projects analytes project
nd Up to 22 Nov 76 1648 21.68 Post merge 2011
Oct 2011 91 1651 18.14 Pre lab Sept 2011 23 270 11.74 merge August 2011 16 184 11.50 Resulting Cost Savings – Urine Bench September 2011 714 Urine Isolates had a Phoenix ID & Sensitivity Cost = £3348.66
October 2011
660 urine isolates had a MALDI ID on 660 urine isolates
Cost of ID - 5p x 2 (double spotting) £66.00
Cost of Disc Sensitivity – 49p x 660 = £323.40 Happy Directorate 86 of these had a Phoenix ID & Sens = £403.34 Manager – Glyn Evans
Total = £792.74
SAVING OF ………………… £2555.92……….. IN ONE MONTH ON ONE BENCH! Where are we now?
• Nov 2011 – RGH & PCH Laboratory Merged = nearly doubled staff and workload. •MALDI-TOF Benchmarking method still working well despite extra workload. •Urines – No other bench top ID kit e.g. Staphaurex used – MALDI ID’s and Disc Sensitivity •Faeces – Only anti-sera for Salmonella & Shigella and Latex test for E.coli 0157 USED – All other ID’s Straight on MALD-TOF. •Resps – All Significant organisms – MALDI and Disc Sensitivity
Unanswered Questions
•Pus – High numbers of Staphylococci. Do we use Staphaurex Kit as a screen and MALDI Positives or straight on MALDI-TOF? •Blood Cultures – Possibility of ID from 4hr cultures – to investigate •Blood Cultures – Do we use Sepsityper Kit for all/some B/C’s? Direct B/C Analysis Using Sepsityper Currently – Day 1 Positive Flagged by BactiAlert Gram Film & Culture & Preliminary Sensitivity Discs Preliminary result given to Consultant Day 2 (24 hrs)– ID using colonial morphology and Commercial ID Kits BD Phoenix panel Day 3 (48hrs) – Confirmed result telephoned to Consultant with Sensitivities
So…….confirmed identification result achieved after a minimum of 48hrs!! Question? Is this good enough?
Bruker Sepsityper Kit & MALDI-TOF – can achieve an ID in <1hr Recent Clinical B/C Examples
Case 1 – NOT USING SEPSITYPER! • B/C Positive 15/10/11 (Sat) after 24hrs, Gram film – Yeasts
• Patient – AP resection in July & developed fistula. Patient ventilated with lines. Patient on Caspofungin.
•Day 2 – Yeast grown & Germ Tube Positive
MALDI ID direct from plate on 17/11/11 – Candida albicans Patient antibiotics changed to Fluconazole.
Question – would this change have been done sooner if Sepsityper was available. Case 2 – USING SEPSITYPER! • B/C Collected on 13/11/11 and Positive on 15/11/11 48hrs • Initial Gram film – GPC, clumps ? Staphylococci • Sepsityper – Staphylococcus capitis before 10am on 15/11/11. • Patient Clinically well and on Augmentin. • No Change of therapy Recent Clinical B/C Examples
Case 3 – USING SEPSITYPER!
• B/C Collected on 14/11/11 @ 14:10 & came up positive on 15/11/11 before 9am - <24hrs
• Initial Gram film – GPC, clumps ? Staphylococci
• Sepsityper – Staphylococcus aureus before 10am on 15/11/11
• Patient Info – Pt had accident 10 days ago, multiple trauma, CRP 460 and Pt on IV Augmentin when result was telephoned. MRSA Neg on 04/11/11.
• Consultant Action – De-escalated antibiotic therapy to Flucloxacillin on 15/11/11.
• Day 2 – S.aureus (Cefoxitin =S) growing on plate and confirmed by Phoenix. Flucloxacillin continued. Clinical Example - CSF
CSF Received on 10/10/11 Patient admitted with 2/52 history headache, vomiting, increased pressure, ? Meningitis. Patient ventilated and admitted to HDU. CSF Count – 398 WBC, 80% Polymorphs. NOS in Gram film. Telephoned to Consultant at 9:30am. Patient was on Teicoplanin as PEN allergic- Not on advice of Microbiologist! Changed to Ceftriaxone, Meropenem, Vancomycin and Aciclovir and samples sent for Meningo and Pneumococcal PCR. 11/10/11- Cultures - Yeast. Original Gram film reviewed, Still NOS. Put straight on MALDI – Cryptococcus neoformans – Surprised but ID within 24hrs Auxocolour – confirmed ID but only after a further 24-72hrs. All other cultures neg and Cryptococcal antigen on CSF confirmed. Therapy – Changed to liposomal amphotericin & flucytosine and history obtained to look for exposure to birds/pigeons and looked for evidence of immunosuppression – None found
MALDI ID – Led the Consultants down a different patient management route. Treatment already followed the antibiotic guidelines as they include an antifungal but MALDI allowed the risk factors to be investigated. AND THEN………………………………………
WE GOT TWO………………………………………
HOW? Problem
• Wednesday - Noticed the MALDI taking a long time to get to vacuum >20 mins but still working. •Engineer came out Thursday afternoon after we had run all samples and arranged a part to be delivered. •Still didn’t work so Bruker arranged a temporary replacement instrument to be delivered on the Friday evening. •Downtime of Instrument was only a day (Friday) •BUT - No downtime of work as target plate spotted and matrix added = stable for up to 5 days. •We had a working Instrument by Saturday so sensitivities read and plate run so no delay in results! EXCELLENT SERVICE FROM BRUKER & I WOULD QUITE LIKE TO KEEP THE TWO!! Thank You & Any Questions