Overview. A day in the life. 8/31/2012. Walkthrough of a typical run. Training, maintenance and QC. Ah ha! moments

Transcription

1 David Winkler MT(ASCP) Microbiology Technical Specialist St. Louis Children s Hospital No financial disclosures Overview Walkthrough of a typical run - Benefits to patient care, cost savings, changes in lab Training, maintenance and QC - Daily, weekly, and as needed issues Ah ha! moments - Examples of useful clinical results A day in the life. Isolated colony Spot on target Add BTS, formic acid Overlay with matrix Perform run Calibrate Place target into MALDI Enter run into computer Print results Enter results in patient workcard 1

2 Where s my results? Results obtained typically within minutes of addition of reagents. Stumbling blocks: - number of isolates spotted - amount of isolate spotted - calibration issues - time of year (humidity) - organism not in library The benefits Can get a reliable identification within minutes instead of overnight Can reduce inventory of biochemical reagents Cost per run Changes to lab procedures St. Louis Children s Hospital 250 bed, tertiary care Children s Hospital Academic Setting Transplant patients Oncology patients Major referral center Reduced turn around time does not equal fewer technologists! 2

3 Test Menu Blood Cultures Stool Cultures Ova and Parasite Analysis Wound Cultures MRSA Screening VRE Screening Surgical specimens Respiratory Cultures Spinal Fluid Cultures Cultures for Fungus Urine Cultures Rapid Strep Testing Rotavirus Cost Savings Example Media Reduction in media TSI Qty 100 = $ boxes at a time TSI Qty 20 = $ boxes at a time Vitek cards GN, GP, YST - $65.00/20 ($3.36 per card) Anaerobe cards - $115.00/20 ($7.19 per card) Cost of materials Matrix cost ($255.00/10 vials) 1 vial = 250μl = $ μl = $.10 BTS cost ($144.00/5 vials) 1 vial = 50 μl = $ μl = $.58 Tips cost ($90.00/960) 1 tip = $.09 Cost per MALDI run For 1 isolate 1 spot BTS ($.58) + 3 spots Matrix ($.30) + 5 tips ($.45) = $ isolates = $1.71 = $.85 per isolate Half the target ( 23 isolates) = $11.67 = $.51 per isolate Full target (47 isolates) = $22.95 = $.49 per isolate Changes in the lab Revisions made to multiple procedures New procedures written Current procedures revised to incorporate MALDI Competency Annual observations Unknowns taken from positive patient blood cultures Proficiency testing Document for CAP surveys use of MALDI when needed No official proficiency testing available Urine cultures Mixed Gram negatives Previous identification = sub both organisms and work up next day MALDI gives result same day of E. coli 3

4 Respiratory cultures Mix of normal flora and possible pathogens Beta colony MALDI ID as Group A strep same day Alpha hemolytic sub with optochin disc and perform bile solubility next day Stool cultures Black colonies from HE Old method sub TSI and LIA, verify with VITEK if possible Salmonella 2 day turn around time MALDI get ID same day Blood cultures Positive blood = Gram stain and critical call to physician with result Previous method- wait for culture to grow to obtain identification New method obtain identification directly from blood broth through extraction Result delivered to physician within hours instead of days Results always verified by comparing to what grows on culture Blood cultures Identifications from positive blood broth done 24/7 Evening and night shift always perform Gram stain immediately and extraction as time permits 2 nd critical call made within 3 hours if reliable identification obtained Extensive communication with critical care units, emergency unit, infectious disease hematology/oncology groups, and residents Extraction method 1. Take 1 ml of blood and add 200μl lysis buffer, centrifuge 2 min. 2. Remove supernatant and add 1 ml wash buffer 3. Vortex and centrifuge 2 min. 4. Remove supernatant and add 300 μl ethanol and 900μl water. 5. Vortex and centrifuge 2 min. 6. Remove all ethanol/water by pipetting and air drying Extraction method 7. Add 70% formic acid, mix, and add equal amounts of acetonitrile. 8. Vortex and centrifuge for 2 min. 9. Spot 1μl of supernatant onto target and let dry 10. Add matrix to spot and perform run. 4

5 Extraction method Approximately min hands on time Kit costs about $ with 50 tests = $5 per extraction QC to be done on each new kit/lot number received Benefit is an identification within an hour instead of an overnight incubation Inventory changes Reduction/elimination in reagents and identification systems TSI, LIA, Rapid test systems, VITEK, Phoenix Backup and verification purposes indole, oxidase, catalse Keep everything on hand for downtime Training Training 2-3 days by representative Super users selected Individuals to understand instrument and then train rest of lab Trained by shifts, usually individually Day shift trained on all aspects Evening and night shift focused more on extractions from blood cultures Training document created for competency MALDI Training Checklist: Name of Trainee: Name of Trainer: Dates of Training: Trainer Tech 1. Understands reagent preparation, storage, documentation of expiration dates, labeling, and safety precautions regarding use of acids. 2. Discussed preparation, shelf life, and handling of both BTS and matrix. 3. Demonstrates correct procedure for spotting a target plate.. 4. Understands the amount (1µl) and location of BTS, matrix and 100% formic acid used on a target for a run. 5. Understands the functions of the 3 software programs used on the Maldi computer. 6. Demonstrates how to create a run and consistently name the file on the computer. 7. Demonstrates the ability to properly load a target into the MALDI and understands that a target should always be kept in the instrument. 8. Demonstrates how to perform both an automatic and a manual calibration and understands when each should be performed with. 9. Describes what steps should be taken if calibration can not be achieved. Who performs the runs? Three potential scenarios: 1. Each bench gets own target to spot and perform run - fewer organisms per target - techs follow themselves throughout whole process - use of more reagents (BTS) - log jam at machine if everyone done at once 10. Demonstrates how a report is generated and printed once a run is completed. 11. Demonstrates how spectra can be collected manually and saved. 12. Demonstrates how saved spectra can be evaluated by MALDI software to achieve identifications. 5

6 Who performs the runs? 2. One target used by all benches: Use of less reagents required All the information in one spot Many organisms on one target Runs may be performed later in the day Results passed around to benches to enter into workcard Who performs the runs? 3. One individual dedicated to performing MALDI Bench techs indicate which organisms should be spotted One person spots, performs run, and enters results Probably preferable for larger labs Person will probably be available only for MALDI that day How do I know this thing is working correctly? Routine QC performed: Daily BTS (Bacterial test standard) Consists of E. coli and two added proteins Used to calibrate each run Used as positive control How do I know it identifies other organisms? Weekly QC: Assortment of organisms: - Pseudomonas aeruginosa - Staphylococcus aureus - Haemophilus influenzae - Clostridium perfringens - Matrix only 6

7 Necessary materials Targets Reagent preparation done weekly: - Organic Solvent, BTS, Matrix Organic solvent = mixture of molecular grade water, Acetonitrile, and Trifluoroacetic acid - total volume of 1 ml. - good for one week from preparation - reconstitute Matrix with 250μl - reconstitute BTS with 50 μl What else is needed? Eppendorf PCR clean pipette tips Formic acid - used for extractions and hacker method Ethanol - used for extractions and cleaning Cleaning method Targets are reusable Cleaned weekly or as needed Process: 1. Overlay target with 70% ethanol for 5 minutes 2. Rinse, wipe clean, and wipe with 70% ethanol 3. Rinse, wipe clean, and overlay with 80% trifluoroacetic acid. (Perform in fume hood) 4. Wipe clean while wearing gloves! 5. Rinse target with deionized water, wipe, and air dry for 15 minutes. 7

8 Other maintenance issues Backup of data done weekly Keep hard copy of runs for 2 months Source cleaning done as needed Filters cleaned as needed Annual preventive maintenance So how has this helped our lab? Scenario #1 Messy cultures Improvement of TAT for enteric cultures - reduce use of LIA and TSI slants - reduce confirmatory Vitek testing Improvement of TAT for anaerobic cultures - reduce use of disk confirmatory testing - reduce need for other anaerobic identification methods - elimination of aerotolerance testing Scenario #2 Phantom menace Positive blood culture early on in midnight tech s shift Gram variable rods seen in Gram stain MALDI run from blood bottle identified as Clostridium perfringens Night shift tech informed blood bench tech first thing the next day and plates examined immediately 8

9 Scenario #2 Phantom menace Isolate put onto MALDI with result of Clostridium perfringens again Culture also grew Pantoea sp. Informed physician of unusual nature of culture where it was discovered patient had gone into septic shock Antibiotic therapy immediately changed after results relayed Scenario #3 Which one is it? Both aerobic and anaerobic bottle from patient flagged as positive MALDI from anaerobic bottle ID as E. coli, aerobic bottle ID as Raoultella ornithinolytica After incubation, culture from anaerobic bottle grew abundant beta hemolytic colonies and few non hemolytic colonies Aerobic culture grew abundant non hemolytic colonies and few beta hemolytic colonies Scenario #3 Which one is it? Beta hemolytic colonies MALDI ID as E. coli Non hemolytic colonies ID as Raoultella ornithinolytica Possible ID of predominant organism from mixed cultures Scenario #4 MALDI knows best Beta-hemolytic colony isolated from a culture Gram positive cocci, catalase pos, Staph latex neg, coagulase neg MALDI id as S. aureus Repeat of biochemicals the same Sent for sequencing, resulted as S. aureus Scenario #5 You called it what? Patient transferred to SLCH from another hospital Blood culture result from other hospital as Streptococcus sp. New blood culture drawn when admitted to SLCH Blood flagged as positive within 12 hours of draw, result from broth as Enterococcus casseliflavus Culture grew organism consistent with ID MALDI result from grown organism = E. casseliflavus 9

10 Scenario #6- I need backup! Anaerobic blood bottle flagged positive Gram positive cocci in clusters No reliable ID from MALDI No growth aerobically, weak growth anaerobically No reliable ID from MALDI from growth, resubbed with aerotolerance test Rapid ANA set up for ID = Staphylococcus saccharolyticus Repeat of MALDI ID as Staphylococcus saccharolyticus just above reporting threshold Benefits to the lab Quick and reliable identifications obtained Low and easy amounts of maintenance required Easier and quicker to identify problem organisms Questions? 10