How Fast Do We Need Results and Technologies That Will Help

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1 How Fast Do We Need Results and Technologies That Will Help Romney Humphries PhD D(ABMM) Clinical Microbiology UCLA David Geffen School of Medicine Los Angeles, CA, USA 1

2 Disclosures Research Funding in past 12 months from: Cepheid, biomerieux, Siemens, Curetis, Qiagen, Cubist, Quidel Honoraria / Advisor to: GenMark, Nanosphere, Multicode, Cubist, Focus Diagnostics

3 How Fast? Use of inappropriate antimicrobials within the first six hours of recognition of septic shock is associated with five-fold higher mortality (52 vs 10.3% survival) Kumar et al 2009 Chest 136:1237

4 How Fast? Average decrease in survival of 7.6% for each hour after the onset of hypotension before initiation of effective antimicrobials Kumar et al 2006 Crit Care Med 34:1589

5 Traditional Blood Culture Workflow Blood Drawn Blood received by laboratory, incubated Culture turns positive Blood subcultured, incubated Physician Notified Unloaded, Gram stain Isolate Identified, Antimicrobial susceptibility testing performed Final Results

6 Traditional Blood Culture Workflow Blood Drawn 2-10 hr Blood received by laboratory, incubated hr Culture turns positive mins -hrs Blood subcultured, incubated Physician Notified Unloaded, Gram stain 4h 24h Isolate Identified, Antimicrobial susceptibility testing performed 18-24hr Final Results mins -hrs hrs

7 Traditional Blood Culture Workflow Blood Drawn 2-10 hr Blood received by laboratory, incubated hr Culture turns positive Blood subcultured, incubated Physician Notified Unloaded, Gram stain Isolate Identified, Antimicrobial susceptibility testing performed Final Results Reduce from days to hours

8 Technologies That Improve Time to Results After Positive Blood Culture Peptide Nucleic Acid Fluorescence in situ Hybridization (PNA-FISH) Matrix-Assisted Laser Desorption Ionization-Time-Of-Flight (MALDI-TOF) Polymerase Chain Reactions (PCRs) Multiplex Systems: Identification and Resistance Determinants

9 PNA-FISH S. aureus /CNS GNR Traffic Light Yeast Traffic Light Peptide nucleic acid (PNA) fluorescent in situ hybridization (FISH) PNA molecules mimic DNA, replacing negative sugar-phosphate backbone with a peptide (can traverse cell membrane) TAT: 30 mins 1.5 hours (QuickFISH vs PNA- FISH) Requires fluorescent microscope To date, identification of organisms only: Coagulase negative Staphylococcus vs S. aureus; E. faecium vs E. faecalis, E. coli, P. aeruginosa vs K. pneumoniae Abbreviations: DNA, deoxyribonucleic acid; TAT, turn around time.

10 MALDI-TOF Matrix-assisted laser desorption ionization time of flight mass spectrometry Two available for clinical use in U.S.: Shimadzu / biomerieux Bruker Microflex $$$ capital purchase (but good ROI) Many labs have adapted to run directly from positive blood culture (LDT) Requires processing of blood (or can use young colonies) Performance varies Abbreviations: ROI, return on investment; LDT, laboratory developed test.

11 hours MALDI TOF from Blood Culture Broth Time to ID by Conventional Methods* 20 minutes by MALDI-TOF, 97% Gram negative ID, 80% Gram positive ID * From positive blood culture. Buchan et al JCM :346-52

12 Performance of MALDI-ToF from Blood Culture Broths Ferreira et al 2011 CMI blood cultures Centrifugation processing, Bruker 86.6% to species, 96.6% to genus among 61 GN 31.8% to species, 64.8% to genus among 239 GP Only 1 / 18 yeast ID Problems: Gram positive ID Yeast Fothergill et al 2013 JCM 259 blood cultures, 28 polymicrobial Filter processing, VitekMS 73% to species 19.7% no ID 2.3% = wrong ID Problems: S. aureus called S. epidermidis A. baumannii called K. pneumoniae C. albicans called M. catarrhalis Martinez et al 2014 JCM 159 blood cultures, 13 polymicrobial Sepsititer processing, Bruker MS 80.1% to species 87.7% to genus Problems: Non- Enterobacteriaceae Polymicrobial infections S. pneumoniae

13 Rapid AST? Neither PNA FISH, nor MALDI TOF yield rapid Antimicrobial Susceptibility Testing (AST) results at present. Highest yield likely if both Identification (ID) and AST results available quickly Strategy: Direct-from-blood broth AST Use positive blood broth to inoculate AST panels. Use MALDI-prepped isolates to inoculate AST panels.

14 Direct-From-Blood Broth AST Machen et al 2014 PLoS One DOI: /journal.pone

15 Performance of Direct AST 1012 microorganism antimicrobial combinations 93.5% agreement with routine Vitek2 results, 2.6% me, 1.7% ME, 1.3% VME Organism ME VME E.coli Cefazolin E. cloaceae Amikacin E.faecium Linezolid, vancomycin P. mirabilis Ampicillin, cefazolin, ceftazidime, ceftriaxone P. aeruginosa SXT Ciprofloxacin S. marcescens Ceftazidime S. aureus Tetracycline, vancomycin Tetracycline S. epidermidis Clindamycin, ertapenem, SXT, vancomcyin, rifampin SXT, vancomycin, gentamicin

16 Significant Decrease in Time to Results GN GP Total Hours from positive culture Machen et al 2014 PLoS One DOI: /journal.pone

17 Direct-from blood broth AST (2) Removed a 6 ml aliquot of blood broth and transferred to vacutainer serum separator tube (SST) Centrifuged 2,000 rpm for 15 minutes Supernatant aspirated and bacteria resuspended in Phoenix ID broth (BD) and processed using autoinoculator (Phoenix AP, BD) Wimmer et al 2012 JCM 50:2452

18 Performance of Direct AST (2) 1,882 organism-antimicrobial tests evaluated (all GNR) Compared to BD Phoenix results from colony growth 98% Categorical agreement; 1.37% me Organism ME VME E.coli Ampicillin, tetracycline, SXT, cefepime K. pneumoniae Tetracycline P. mirabilis Cefazolin (2), cefuroxime P. aeruginosa Imipenem, piperacillintazobactam aztreonam

19 Multiplex Detection Verigene System, Nanosphere Five minutes hands on time; two hours to results. FilmArray System, Biofire (biomerieux) Two minutes hands on time; one hour to results. Numerous others in development

20 Nanosphere Verigene Organisms S. aureus S. epidermidis S. lugdunensis S. anginosus group S. agalactiae S. pneumoniae S. pyogenes E. faecalis E. faecium Staphylococcus spp Streptococcus spp Listeria spp. Resistance Determinants meca vana vanb

21 Nanosphere Verigene Organisms E. coli K.pneumoniae K. oxytoca P. aeruginosa S. marcescens Acinetobacter spp. Proteus spp. Citrobacter spp. Enterobacter spp. Resistance Determinants KPC NDM CTX-M VIM IMP OXA

22 Performance of Verigene Gram Positives (Samuel et al 2013 JCM 51:1188) 92% concordance overall for ID, 96% for resistance Lower if polymicrobial: 76 & 84%, respectively Gram Positives (Buchan et al 2013 PLOS Medicine) 1252 GP blood cultures, sensitivity ranged from 92.6% - 100% sensitive and 95.4% - 100% specific Polymicrobial cultures: 71.6% concordance Gram Negatives (Tojo et al ASM 2013) 206 simulated specimens, 98% concordance K. pneumoniae, 87.5%

23 FilmArray Gram Positives Gram Negatives Yeast S. aureus S. agalactiae S. pneumoniae S. pyogenes Enterococcus Staphylococcus Streptococcus spp Listeria monocytogenes meca vana/b KPC A.baumannii H.influenzae N.meningitidis P.aeruginosa Enterobacteriaceae E.cloacae E.coli K.pneumoniae K.oxytoca Proteus S.marcescens C.albicans C.glabrata C.krusei C.parapsilosis C.tropicalis

24 Performance of the FilmArray Rand and Delano DMID : positive blood cultures: 18, no ID (not in panel) 98% to genus, 100% to species Altun et al JCM : positive blood cultures 13, no ID (not in panel) One missed identification - Coagulase negative Staphylococcus (CoNS) Six samples, an additional organism detected (Enterococci, C. albicans); 2 FP meca detections Polymicrobial, all targets present identified in 71%

25 Bypass the Culture? Yield of blood cultures is low (~13%) Significantly impacted by: Volume of blood drawn Antimicrobial administration prior to draw (can be mitigated by resins, see Zadroga et al 2012 CID 56) MALDI ID Traditional ID Direct AST Traditional AST Hours from blood collection

26 Traditional Blood Culture Workflow Blood Drawn Blood received by lab, incubated Culture turns + Blood subcultured, incubated 1-2 hr Physician Notified Unloaded, Gram stain Isolate Identified, AST performed Final Results

27 Direct Detection From Blood Avoid one-two days for a blood culture to turn positive. Strategies include: Broad-range PCR (16S or 23S rrna genes, 18S rrna gene of fungi) None available in U.S. at present, several in Europe / under development Examples: SeptiFast system (Roche) SepsiTest (Molzym) T2 magnetic resonance system Curetis Unyvero Accelerate Diagnostics

28 SeptiFast Detects and identifies 25 most common bacteria & fungi that cause BSI Targets ITS sequences 300 CFU/mL or less 6 hour TAT Gram Positives Gram Negatives Yeast S. aureus Streptococcus spp S. pneumoniae E.faecium E.faecalis CoNS A.baumannii S.maltophilia P.aeruginosa E.cloacae/aerogenes E.coli K.pneumoniae K.oxytoca P.mirabilis S.marcescens C.albicans C.glabrata C.krusei C.parapsilosis C.tropicalis A.fumigatus

29 Performance of Multiplex PCR From Whole Blood Reinhart et al CMR : % of culture positive results are not detected by PCR (even if covered by primer pair) Bacteremia <300 cfu/ml PCR inhibitors In many instances, may have culturenegative, PCR positive results DNAemia appears to be clinically significant in limited studies (see for instance Louie et al 2008 Crit Care Med, Bloos 2010 Intensive Care Med) Adjunct to, not replacement of, blood cultures

30 SepsiTest Based on universal PCR and sequence identification Enriches and isolates bacterial and fungal DNA Depletes human DNA (MolYsis technology) Four hour TAT to presence of bacteria /fungi Limited data on performance, One study evaluated 66 patients, o 26 negative, 8 positive concordant results with culture o 25 patients culture-negative but PCR-positive (4 questionable)

31 T2 Biosystems Magnetic resonance-based technology that measures water molecules reacting in the presence of magnetic fields Use particles with magnetic properties that bind to targets & enhance the resonance signals T2 Candida in development: five species of Candida 3.5 hours to detection 1 CFU/mL LOD

32 Accelerate Diagnostics Direct-from-specimen ID (One hour) and AST (Five hours) <10 4 CFU/mL Detect 90% Gram positive and Gram negative bacteria Uses automated microscopy to evaluate live cell suspensions Algorithm for ID includes cell morphology, growth rate, and geometric growth pattern, etc. AST evaluated by observing growth in presence of antimicrobial & applying computed growth probability scores

33 Example: Klebsiella Pneumoniae Carbapenemase (KPC) Burnham et al JCM In press

34 Curetis Unyvero Only % of patients with sepsis have an organism isolated from blood cultures. Unyvero LRT assay detects 14 bacterial pathogens, Pneumocystis, and 20 resistance determinants. Less than four hours TAT

35 Rapid Methods Summary Many technologies available from positive blood cultures Many in development for direct-from-blood Most are expensive (i.e. $50-$100 per specimen) Limits use to large academic centers However, smaller labs can still focus on reducing time to results: Focus on Gram Stain TAT, use of direct-from-broth methods, PBP2a testing, etc.

36 Example: Gram Stain TAT Barenfanger et al AJCP P= Mortality LOS 5 0 <1 hr >=1 hr

37 Thank you! Questions? 37