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 [email protected]
1
Disclosures
Research Funding in past 12 months from:
• Cepheid, bioMerieux, Siemens, Curetis, Qiagen, Cubist, Quidel
Honoraria / Advisor to:
• GenMark, Nanosphere, Multicode, Cubist, Focus Diagnostics
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
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
Traditional Blood Culture Workflow
Blood Drawn Blood received by
laboratory, incubated
Culture turns positive
Unloaded, Gram stain
Physician Notified Blood subcultured,
incubated
Isolate Identified, Antimicrobial susceptibility
testing performed
Final Results
Traditional Blood Culture Workflow
Blood Drawn Blood received by
laboratory, incubated
Culture turns positive
Unloaded, Gram stain
Physician Notified Blood subcultured,
incubated
Isolate Identified, Antimicrobial susceptibility
testing performed
Final Results
2-10 hr
14- 24 hr
mins -hrs
mins -hrs 4h –
24h
18- 24hr
38 – 82 hrs
Traditional Blood Culture Workflow
Blood Drawn Blood received by
laboratory, incubated
Culture turns positive
Unloaded, Gram stain
Physician Notified Blood subcultured,
incubated
Isolate Identified, Antimicrobial susceptibility
testing performed
Final Results
2-10 hr
14- 24 hr
Reduce from days to hours
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
PNA-FISH
• 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
S. aureus /CNS
GNR Traffic Light
Yeast Traffic Light
Abbreviations: DNA, deoxyribonucleic acid; TAT, turn around time.
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.
MALDI TOF from Blood Culture Broth
0
20
40
60
80
100
120
140
ho
urs
Time to ID by Conventional Methods*
* From positive blood culture. Buchan et al JCM 2012 50:346-52
20 minutes by MALDI-TOF, 97% Gram negative ID, 80% Gram positive ID
Performance of MALDI-ToF from
Blood Culture Broths
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
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
• 318 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
Ferreira et al 2011 CMI 17
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.
Performance of Direct AST
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
1012 microorganism – antimicrobial combinations 93.5% agreement with routine Vitek2 results, 2.6% mE, 1.7% ME, 1.3% VME
Significant Decrease in Time to Results
GN GP Total
0 24 48 72 96 Hours from positive culture
Machen et al 2014 PLoS One DOI: 10.1371/journal.pone.0087870
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
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, piperacillin-tazobactam
aztreonam
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
Nanosphere Verigene
Organisms Resistance Determinants
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.
mecA vanA vanB
Nanosphere Verigene
Organisms Resistance Determinants
E. coli K.pneumoniae K. oxytoca P. aeruginosa S. marcescens Acinetobacter spp. Proteus spp. Citrobacter spp. Enterobacter spp.
KPC NDM CTX-M VIM IMP OXA
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%
FilmArray
Gram Positives Gram Negatives Yeast
S. aureus S. agalactiae S. pneumoniae S. pyogenes Enterococcus Staphylococcus Streptococcus spp Listeria monocytogenes
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
mecA vanA/B KPC
Performance of the FilmArray
• Rand and Delano DMID 2014 79:293-7
– 151 positive blood cultures:
• 18, no ID (not in panel)
• 98% to genus, 100% to species
• Altun et al JCM 2013 51:4130
– 206 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%
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)
0 24 48 72 96 120 144 Hours from blood collection
MALDI ID Traditional ID Direct AST Traditional AST
Traditional Blood Culture Workflow
Blood Drawn Blood received
by lab, incubated
Culture turns +
Unloaded, Gram stain
Physician Notified
Blood subcultured,
incubated
Isolate Identified, AST
performed Final Results
1-2 hr
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
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
Performance of Multiplex PCR
From Whole Blood
• 20-30% 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 culture-negative, 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 Reinhart et al CMR 2012 25:609
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)
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
Accelerate Diagnostics
• Direct-from-specimen ID (One hour) and AST (Five hours)
• <104 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
Curetis Unyvero
• Only 34.2-35.2% 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
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.
Example: Gram Stain TAT
Barenfanger et al AJCP 2008
0
5
10
15
20
25
<1 hr >=1 hr
Mortality
LOS
P=0.03