Rapid Rewards. Identification from Positive Blood Cultures
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1 Rapid Rewards Identification from Positive Blood Cultures Carol Young, MT(ASCP) University of Michigan Health System Clinical Microbiology Laboratory
2 Disclosures Carol Young BioRad (chromogenic media)
3 University of Michigan Clinical Microbiology Laboratory
4 My first blood culture experience 1974 BACTEC 301 The third generation, BACTEC 301 Manually operated carbon-14 based instrument handled one vial at a time Over 500 units were sold to small- and mediumsized hospitals at a price of less than $4, BACTEC Homemade broths plus pour plate BD blood broth (vacutainer) 1988 Isolator + BD vacutainer broth 1993 Continuous monitor system (Organon Teknika)
5 Time to results Blood culture collection Positive culture Gram stain Identification setup Susceptibilities Organism ID Incubation Incubation Identification incubation 0h 12-48h 12-24h 6-24h Patient care team sees ID and susceptibility results?? hours Targeted therapy Decrease morbidity and mortality Improve patient outcomes Reduce hospital acquired infections
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7 Factors to Consider Blood culture system/bottle type Molecular instrumentation Personnel resources & competency Complexity of test Hands on time to perform test Time to result Is the result actionable Antibiotic Stewardship Team
8 Positive Blood Cultures UMICH January-March 2014 (n=1007) Organism n Coagulase Negative Staphylococcus 185 Enterococcus faecium 142 Staphylococcus aureus 107 Yeast 81 Escherichia coli 43 Enterococcus faecalis 33 Klebsiella pneumoniae 31 Enterobacter cloacae 26
9 Evolving Technology 1970-Now Gram stain/quellung Direct tube coagulase Spin, pellets identification/susceptibility Direct bacterial antigen detection 2000-Now Chromogenic media PNA-FISH/QuickFISH Molecular tests directly on positive blood culture bottles MALDI - TOF Future Direct molecular from blood
10 Gram positive cocci, clusters Is it Staphylococcus aureus? Direct Coagulase Test Discs Chromogenic media Antigen detection PNA-FISH or QuickFISH Molecular MALDI-TOF Is it MRSA? No Maybe Yes No No under review Yes Maybe
11 Direct Coagulase Test Method 1. Add 2-3 drops of blood-broth sample in 0.5mL rabbit plasma. 2. Incubate in 36`C heat block and read up to 4 hours. 3. Negative tubes should incubate overnight at RT. Organism* VersaTREK (319) *Identification by Staphaurex (Remel) Time to Positive Positive (%) Negative (%) S. aureus (96) 1 hr 30 (31.3) 2 hr 35 (36.5) 3 hr 9 (9.4) 4 hr 8 (8.3) overnight 8 (8.3) 6 (6.3) Not S. aureus (223) Evaluation of the BinaxNOW Staphylococcus aureus Test for Rapid Identification of Gram-Positive Cocci from VersaTREK Blood Culture Bottles. Dhiman, et al. J Clin Microbio. Sep 2013: 51(9):
12 Discs GPC, clusters: bacitracin and oxacillin GPC, chains or pairs: optochin and vancomycin Read at hours: bacitracin >9mm not Staphylococcus optochin >14mm Streptococcus pneumoniae oxacillin R MRSE or MRSA vancomycin R VRE, usually E. faecium vancomycin-dependent Enterococcus Pediococcus, Leuconostoc
13 Chromogenic Media Vancomycin Resistant Enterococus (VRE) Staphylococcus aureus MRSA Candida
14 Chromogenic Media Alternative Use for Spectra MRSA Chromogenic Agar in Detection of Methicillin- Resistant Staphylococcus aureus from Positive Blood Cultures Spectra MRSA medium was tested at 24 and 48 h using 629 positive blood cultures that exhibited Gram-positive cocci on initial microscopic examination. TABLE 1. Combined clinical trial site data for sensitivity, specificity, PPV, and NPV analysis of Spectra MRSA agar for the detection of MRSA a Incubation period (h) No. of: TP b FP TN FN Sensitivity (%) Specificity (%) PPV (%) NPV (%) b A true positive (TP) is defined as a denim blue colony on Spectra MRSA that was identified as MRSA by a positive Oxoid PBP2 test and an oxacillin MIC of 4 μg/ml. Strains of S. aureus with an oxacillin MIC of 4 to 6 μg/ml that tested PBP2 negative were also classified as TP. Jess F Peterson, et al, J Clin Microbiol. Jun 2010; 48(6):
15 Chromogenic Media 1. Write time on plate 2. Incubate non-co2 3. Early read Clinical Outcomes with Rapid Detection of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Routine Blood Cultures Nicolsen, J. Clin. Microbiol. December 2013 vol. 51 no Media: CHROMagar MRSA II Read Time: 12 hrs SaSelect MRSASelect Rapid culture-based detection of MRSA and MSSA plays a role in antimicrobial stewardship by decreasing the time that it takes to place patients on targeted antimicrobial therapy.
16 Antigen Detection Blood Culture Latex agglutination Blood broth supernatant or Direct Immulex S. pneumoniae OMNI Lateral flow BinaxNow Staphylococcus aureus N=319 BNSA + BNSA - Positive (96) 92 4 S. pneumoniae Negative (223) Enterococcus 5 seconds Sensitivity 95.8% Specificity 99.6% VersaTREK 30 minutes Reference Method: BD Biosciences latex agglutination Quick detection of 92 S. pneumoniae serotypes directly from a positive blood culture or a pure culture Evaluation of the BinaxNOW Staphylococcus aureus Test for Rapid Identification of Gram-Positive Cocci from VersaTREK Blood Culture Bottles. Dhiman, et al. J Clin Microbio. Sep 2013: 51(9): Two posters at International Pneumococcus Congress. 2014, Hyderabad, India
17 Molecular and MALDI-TOF Best fit for your lab Workflow Cost Time to result Targets: ID/Resistance mechanisms
18 PNA FISH Rapid, Simple & Easy Protocol Prepare Smear Hybridize Wash Examine 5-20 min.* 30 min. 30 min. View Results Add drop of Fixation Solution Add drop from BC+ Fix bacteria/yeast onto slide Add PNA Probe Probe enters cells and binds to target rrna sequence, if present Immerse slide in Wash Solution Unbound and excess PNA Probe removed from cells and slide Fluorescence microscopy using 60x or 100x oil objective Target bacteria/yeast fluoresce Heat Methanol, or Flame fixation Turn Around Time 90 min. Hands-On Time = 10 min. Please see package insert for complete instructions for use *Dependent on fixation method PN1870A
19 AdvanDx PNA-FISH S. aureus/cns E. faecalis/oe GNR: P. aeruginosa / E. coli GNR Traffic Light adds K. pneumoniae QuickFISH Faster hybridization No wash step On-board controls 20 minutes <5 minutes hands on time C. albicans/c. glabrata Yeast Traffic Light QuickFISH Green Red Direct Coag Test 4/24 hr S. aureus (35) 35 28/31 CNS (141) 139 Other (3) 0 0 E. Carretto, et al. JCM 51(1)
20 Impact of the Addition of Peptide Nucleic Acid Fluorescence in Situ Hybridization (PNA FISH) to Fluconazole Susceptibility Testing on the Time to Appropriate and Optimal Antifungal (AF) Therapy for Candida Blood Stream Infections (BSIs). IDSA 2010 Peggy L. Carver, PharmD, Melissa A. Carroll, PharmD; Carol Young, MT; Peggy Mahlmeister, MLT, University of Michigan Health System, Ann Arbor, MI Time (days) Sendout FLU/No FISH FLU Suscept/No FISH FLU Suscept/PNA FISH Appropriate AF therapy 2.5 ± ± ± 1.6 Optimal AF therapy 2.7 ± ± ± BC alert to MD 2.4 ± ± ± 1.3 PNA FISH results ND ND 3.3 ± 1.8 Candida speciation 4.6 ± ± ± 2.2 FLU susceptibility results 17.6 ± 15.1** 6.8 ± ± 2.7 Why did PNA FISH fail to decrease time to appropriate or optimal antifungal therapy:? Many patients died before PNA FISH testing could be performed. In some cases, clinicians appear to have ignored PNA FISH test results; they appeared to wait for the results of fluconazole susceptibility testing. Conclusions: Despite an time to fluconazole susceptibility test results, the addition of FLU susceptibility testing, alone or + PNA FISH testing, did NOT significantly appropriate AF therapy or optimal AF therapy for Candida BSIs overall.
21 GeneXpert real-time PCR GeneXpert MSSA/MRSA from blood cultures
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23 Cepheid Xpert MRSA/SA Blood Culture Assay Intended use: Performed on the GeneXpert Instrument Systems Qualitative in vitro diagnostic test Detection of Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) DNA directly from positive blood cultures. The assay utilizes automated real-time polymerase chain reaction (PCR) for the amplification of MRSA/SA specific DNA targets and fluorogenic target-specific hybridization probes for the real-time detection of the amplified DNA. The assay is performed directly on positive blood culture specimens from that are determined by Gram Stain as Gram-Positive Cocci in Clusters (GPCC) or as Gram-Positive Cocci in singles (GPC). BD BACTEC Plus Aerobic/F BacT/ALERT SA (Standard Aerobic) VersaTREK REDOX 1R (aerobic) Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing or for epidemiological typing. The Cepheid Xpert MRSA/SA Blood Culture Assay is not intended to monitor treatment for MRSA/SA infections.
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27 The Verigene System Instruments & Consumables Instruments Verigene Reader Verigene Processor SP Lab Consumables 1. Verigene Test Cartridge 2. Verigene Tip Holder Assembly 3. Verigene Extraction Tray 4. Verigene Utility Tray
28 Verigene Blood Culture Tests Intended Use Verigene BC-GP and BC-GN are multiplexed in vitro diagnostic tests for the detection and identification of pathogenic gram-positive and gram-negative bacteria Verigene BC-GP and BC-GN are indicated for use in conjunction with other clinical and laboratory findings such as culture and is not to be used to monitor bloodstream infections Sub-culturing is necessary for antimicrobial susceptibility testing, identification of organisms non-detected by BC-GP or BC-GN, differentiation of mixed growth, and association of resistance marker or epidemiological typing Genus Resistance Gram-Positive Blood Culture (BC-GP) Test Staphylococcus spp. Streptococcus spp. Listeria spp. meca 1 vana 2 vanb 2 Species Staphylococcus aureus Staphylococcus epidermidis Staphylococcus lugdunensis Streptococcus pneumoniae Streptococcus anginosus Group Streptococcus agalactiae (GBS) Streptococcus pyogenes (GAS) Enterococcus faecalis Enterococcus faecium 1. The meca resistance marker is only tested for if S. epidermidis and/or S. aureus is Detected 2. The vana/vanb resistance marker is only tested for if E. faecalis and/or E. faecium is Detected Genus Resistance Gram-Negative Blood Culture (BC-GN) Test Acinetobacter spp. Citrobacter spp. Enterobacter spp. Proteus spp. CTX-M (bla CTX-M ) KPC (bla KPC ) NDM (bla NDM ) VIM (bla VIM ) IMP (bla IMP ) OXA (bla OXA ) Species Escherichia coli 3 Klebsiella pneumoniae Klebsiella oxytoca Pseudomonas aeruginosa 3. BC-GN will not distinguish Escherichia coli from Shigella spp. (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei)
29 Consumable Differences Consumable BC-GP BC-GN Difference Extraction Tray 2 8 C 2 8 C Sample Loading Well location Sample Volume Sample Loading Well 350 µl of Gram Positive Blood Culture Media Sample Loading Well 700 µl of Gram Negative Blood Culture Media Tip Holder Assembly Utility Tray <8 C 2 30 C -20 C None Storage conditions. Contains tube with PC pellet (BC-GP) vs. no tube (BC-GN) Test Cartridge 2 8 C None Sample Well Caps N/A 2 30 C Only available for BC-GN
30 The Verigene System Hybridization Technology Overview
31 FilmArray vs Verigene D-106 ICAAC 2013 "Nanosphere Verigene v BioFire FilmArray for Rapid ID of Gram-Positive Cocci in Blood Cultures," C.J. Sailey et al.; Arkansas Children's Hospital and the University of Arkansas, Ark. Bactec (n=80) g+c,cl BioFire FilmArray BCID Nanosphere Verigene BC-GP Hands on time 5 minutes 5 minutes Time to result 1 hour 2.3 hours Sensitivity 96% 100% Specificity 97.5% 100% PPV 99% 100% Limitations Cross reactivity with Enterococcus Staphylococcus genus only, except S. aureus meca: S. aureus S. epidermidis Conclusion: Both of these tests offer a viable solution for rapid identification of GPC from positive blood cultures.
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33 Gram Positive (Evaluation of FilmArray and Verigene Systems for Rapid Identification of Positive Blood Cultures) FilmArray (BioFire) Verigene (Nanosphere) Correct Incorrect Correct Incorrect S. aureus (32) meca + (13) meca (19) Coag Staph (32) Enterococcus (9) 9 9 vana + (4) vana (5) 4 5 Streptoococcus 9 9 TOTAL (82) identifiable GP 77 (94) 5 (6) (100) Resistance Markers 40 (98%) 1 (2%) 41 (100%)
34 Gram Negative (Evaluation of FilmArray and Verigene Systems for Rapid Identification of Positive Blood Cultures) FilmArray (BioFire) Enterobacteriaceae (23) 23 Enterobacter sp. (1) E. cloacae (1) 1 Verigene (Nanosphere RUO) E. coli (11) K. pneumoniae (6) 6 6 K. oxytoca (1) 1 1 S. marcescens (2) Citrobacter (1) 1 P. aeruginosa (4) Not identified Misidentified Identifiable GN (27) 27 (100%) 25 (96%) 1 Resistance Markers KPC(1) CTX-M (1) 1 1 1
35 MALDI-TOF Matrix Assisted Laser Desorption Ionization Time of Flight Mass spectrometry
36 MALDI-TOF Two systems in the US Biotyper (Bruker) Vitek MS (biomérieux) Studies for many organism groups aerobes anaerobes fungi (both yeast and moulds) mycobacteria Nocardia Direct detection from positive blood cultures and urine Future differentiation of resistant vs. susceptible isolates
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38 MALDI-TOF 1. Harvest cells from positive blood broth, using SepsiTyper Medium time from BC+ to extraction was 4.3 hr Spot target plate Hopkins, Brown, UA ASM Poster, 2012 Bactec n=226 TREK n=155 n=347 SepsiTyper (%) Agree 275 (79) Discordant 9 (3) 2. Lysis filtration A Fothergill, J Clin Micrbiol. March In house saponin C Meex, J Med Microbiol, Nov 2012 No ID 16 (5) Insufficient protein 47 (13) Mixed, excluded 34
39 MALDI-TOF from early incubation cultures First timers plate rack Mean Time to species ID <2 hrs <4 hrs <8 hrs <12 hrs GPC (86) 5.9 hrs 1.2% 18.6% 98.5% 98.8% GPC (34), extracted 3.1 hrs GNR (42) 2.0 hrs 76.2% 95.2% 97.6% 97.6% Rapid Identification of Microorganism from Positive Blood Cultures by MALDI-TOF Mass Spectrometry Subsequent to Very Short-Term Incubation on Solid Media Idelevich EA, et al. Clin Microbiol Infect, 2014 April 3 Limitations: mixed cultures
40 Questions to ask: How will this affect our workflow? What personnel is required for testing What instrument(s) do I have available for testing? Real time vs. batching How many instruments will we need for real-time testing? Availability of verification/validation materials? Is the result actionable at my facility ID/AST/Pharmacy System FilmArray BCID Verigene GP, GN PNA-FISH QuickFISH Xpert MRSA/SA BC MALDI-TOF Broth/early plate Hands on time 5-10 min 5-10 min 15 min 5-10 min SepsiTyper: 45 min / 5 min Early plate: 2-6 hrs Time to result 1 hr hrs 30 min 1 hr 1 hour / 5 min Complexity Moderate Moderate High Moderate High Cost Instrument Reagent $$ $$$$ $$ $$$ $ $$ Bottle types Non-charcoal All Non-charcoal Product insert Manufacturer/None QC/Controls On board On board On board On board Each target plate $$ $$$ $$$ $
41 System Identification Gram Positive BioFire Nanosphere AdvanDx Cepheid biomerieux Bruker FilmArray BCID Staphylococcus (1) Streptococcus (3) Enterococcus (1) Listeria Verigene GP, GN Staphylococcus (3) Streptococcus (4) Enterococcus (2) Listeria PNA-FISH QuickFISH Staphylococcus (2) Enterococcus faecalis (other Enterococci) Xpert MRSA/SA BC Staphylococcus (2) MALDI-TOF Species in the respective libraries Gram Negative Enterobacteriaceae (6) E. coli K. pneumoniae K. oxytoca Proteus spp. Enterobacter spp. Serratia marcescens P. aeruginosa A. baumannii H. influenzae N. meningitidis E. coli K. pneumoniae K. oxytoca Proteus spp. Citrobacter spp. Enterobacter spp. P. aeruginosa Acinetobacter spp. E. coli K. pneumoniae P. aeruginosa Yeast Candida (5) In development (8) PNA (5) Quick pending FDA submission Resistance meca vana, vanb KPC meca vana, van B KPC, NDM,CTX-M, VIM, IMP, OXA meca XpressFISH for S. aureus (FDA under review) meca, SCCmec In development
42 Clinical Infectious Diseases, 2013; 57(9): Antibiotic Stewardship Team Interventions The AST made a total of 210 interventions 189 (90.4%) interventions were accepted 81 (38.6%) 54 (25.7%) 75 (35.7%) Time of Intervention (N=210) Gram stain Organism ID Sensitivities 68 (32.4%) Type of Intervention (N=210) 17 (8.1%) 53 (25.2%) 72 (34.3%) Initiated or Broadened Therapy Narrowed Coverage (based on organism) Narrowed Coverage (excess coverage stopped) Other
43 UMICH Workflow Support Technologist (7-11 am, afternoons, midnights) Blood Technologist (7am-3pm) Blood Culture Instrument Signals Read chromogenic Perform rapid tests Unload bottle(s), make slide and subculture ID/AST Perform MALDI Notify physician Gram stain slide Read plates.discs Read direct coags
44 Number of bottles Best Fit for Lab Manufacturer/bottle types Molecular instrumentation Verification/Validation How many do I have vs need for real time testing? Time to result Personnel staffing requirements Day 1 Day 2 Day 3 Day 4 Real time review/action on results real time?
45 Bruker SepsiTyper vs BioFire BCID (University of Michigan CLS student presentation at Mi ASCLS April 2014) Traditional Identification (n=53) MALDI Bruker SepsiTyper BioFire BCID Staphylococcus aureus 6 9 Comments Coagulase negative Staph 6 8 BioFire: genus only, not S. aureus meca positive/negative N/A % concordance with cefoxitin screen on Vitek 2 Streptocococus 3 6 BioFire reports genus Streptococcus species pneumoniae, pyogenes, agalactiae Enterococcus 6 6 BioFire: genus only, except E. faecalis MALDI: species vana/b N/A 6 E. faecalis = negative E. faecium = positive Gram positive rods 4/5 not in database MALDI did not get Bacillus Escherichia coli 6 6 Klebsiella pneumoniae 6 6 Other Enterobacteriaceae 3 3 BioFire: genus only MALDI: speciates Candida 3 4 Species confirmed MALDI missed 2 nd organism
46 Summary AST: Treatment of culture proven invasive infections. (CDC) Bloodstream infections present good opportunities for interventions to improve antibiotic use because they are easily identified from microbiology results. The culture and susceptibility testing often provides information needed to tailor antibiotics or discontinue them due to growth of contaminants. We have a lot to learn about these new systems and opportunities to partner with our Antibiotic Stewardship Team to decrease the time to actionable result to improve patient care with targeted therapy. Challenge ourselves while being aware of the limitations of the various systems as we make these investments in finding the best fit for our laboratories.
47 Thank You Manufacturers who have provided me with information and/or test material UMICH laboratory staff for supporting my endeavors
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