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 rhumphries@mednet.ucla.edu

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.

Direct-From-Blood Broth AST

Machen et al 2014 PLoS One DOI: 10.1371/journal.pone.0087870

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

Example: Klebsiella Pneumoniae

Carbapenemase (KPC)

Burnham et al JCM In press

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

Thank you!

Questions?

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