Direct Identification of Microbes in Positive Blood Cultures

Direct Identification of Microbes
in Positive Blood Cultures
APHL Teleconference
6/18/2013
Donna M. Wolk, MHA, Ph.D., D(ABMM)
System Director, Clinical Microbiology
Department of Laboratory Medicine, Geisinger Health Systems
Professor, College of Pharmacy/Science, Wilkes University
1
Faculty Disclosure
The following disclosures of personal financial relationships with commercial interests within the
last 12 months as relative to this presentation
Research Contracts: Accelerate, AdvanDx, Biofire, MDC, MicroPhage
Speaker: AdvanDx, Qiagen
Consultant: Abbott Molecular, Accelerate, Cepheid
2
Objectives
1. Identify the key laboratory challenges for
detection of bloodstream infections
2. Describe and discuss strategies to improve
the identification and treatment of
bloodstream pathogens
3
3
Laboratory Challenges for
Detection of Bloodstream Infection
(BSI)
Laboratories play a key role.
4
Gradient of Disease
1. Bacteremia/Bloodstream Infection (BSI)
2. Systemic Inflammatory Response Syndrome
(SIRS)
3. Sepsis: SIRS plus a culture-documented infection
4. Severe Sepsis: Sepsis plus >1 organ dysfunction
5. Septic Shock
•
•
Hypotension (despite fluid resuscitation) plus
Hypoperfusion (may include, but are not limited to lactic
acidosis, oliguria, or an acute alteration in mental status.)
6. Death
Infection
5
SIRS
Sepsis
Severe Sepsis
Septic Shock
Death
BSI & Sepsis Can be Difficult to Diagnose
Wolk, 2010, Clin. Microbiol. Newsletter Vol. 32: and 32:6
Bacteremia
Fungemia,
Parasitemia,
Viremia,
Other
Infection
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SEPSIS
Burns,
Trauma,
Pancreatitis,
Other
SIRS
Severe
Inflammatory
Response
Syndrome
BSIs are Common and Increasing
• Sepsis is 10th leading
cause of death in U.S.2
• Condition w/ highest
increase (2001-2007)1
– 97% more
$12.3B
675,000
342,500
$4.5B
• Mortality
– Bacteremia/Candidemia 1
 14% (community)
 34% (HAI)
 Higher in fragile pops.
7
1.AHRQ News and Numbers, June 9, 2010. Agency
for Healthcare Research and Quality, Rockville, MD.
http://www.ahrq.gov/news/nn/nn060910.htm
2. Angus DC, et al. Crit Care Med. 2001;29(7):1303-1310
3.Diekema et al. J Clin Microbiol. 2003 Aug;41(8):3655-60
4. Angus DC, et al. Crit Care Med. 2001;29(7):1303-1310.
Disease Progresses Quickly
Improvements are Needed
Endothelial Inflammation
Pathogen
Infection
Dysfunction
Host
HOST
Response
RESPONSE
Coagulation/
Other
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Fibrinolysis
1
• Loss of homeostasis
2
• Organ dysfunction
3
• Death
8
“UNTIL A CURE FOR SEPSIS IS FOUND,
EARLY DETECTION IS THE SUREST HOPE FOR SURVIVAL”
…THE SEPSIS ALLIANCE
Rapid Antibiotic Therapy (Rx) Saves Lives
Kumar A. et al., Crit Care Med 2006, 34:1286
Dellinger RP, et al. 2008. Crit Care Med 2008;36:296‐327.
Jaeschke RZ, et al Pol Arch Med 2008;118:92‐95.
Kollef MH, et al Chest 1999;115:462‐474.
Garnacho‐Montero J, et al, Crit Care Med 2003;31:2742‐2751.
Valles J, et al, J Infect 2008;56:27‐34.
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Broad-spectrum Rx May Require De-escalation
• Re-evaluate daily to…
–Optimize efficacy
–Avoid toxicity
–Prevent resistance
–Minimize costs
• Discontinue broad spectrum Rx w/in 3 - 5 d
Sharma, S. and Kumar, A. 2008 Clin Chest Med, 29(4)
Deresinski, S., CID 2007:45 (Suppl 3),S177
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Antiobiotic Vigilance
Actionable Results
Microbiology Laboratory, Pharmacy, Physicians
Focus on Antimicrobial De-escalation
and Antimicrobial Stewardship
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Dx
Microbiology Drives
Antibiotic Deescalation
Results to ID Pharmacy
Rapid Testing
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Tier One
Rapid Testing on Positive
Blood Culture Broth
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Mortality increased by 19.2% if > 1 hr , (Ave. 3.3 hr) n = 99 (p < 0.05)
14
Barenfanger et al. Am J Clin Pathol. 2008 Dec;130(6):870‐6
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Blood Culture Gram’s Stain Dilemmas?
15
Gram-positive cocci
in clusters (GPCC)
Gram-pos. cocci pairs
& chains (GPCPC)
Gram-negative rods
(GNR)
Yeast
S. aureus vs CoNS
Infection vs
Contamination
E. faecalis
E. faecium
VRE
S. pneumoniae
P. aeruginosa
K. pneumoniae
E. coli
Other GNR
C. albicans
C. parapsilosis
C. glabrata
Other Candida
MIC Creep
Vanc vs High Dose
Vanc
Daptomycin
Linezolid
Ampicillin
Ceftriaxone
Vancomycin
Daptomycin
Linezolid
Pseudomonal Rx
Ceftriaxone
Cefepime
Pip-Tazo
Imipenem
Ertapenem
Fluconazole IV
Fluconazole PO
Echinocandin
Amphotericin B
Courtesy AdvanDx
Rapid Phenotypic Methods
Most are Pre-CLIA
Few publications statistically
address impact
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16
S. aureus ID, Direct Tube Coagulase
Test (DTCT)
• Positive Agreement (PA)
• 4hr DTCT unless specified
•
•
•
•
•
64% PA, (Qian et al, 2007, J Clin Microbiol)
85% PA, (Chapin et al, 2003 J Clin Microbiol)
92% PA, 2hr. ( Cook et al, 1997, J Clin Path)
96% PA, (Sterm et al 2008, Clin Microbiol Infect)
96.8% PA (95% CI 81.5 - 99.8) n = 47,(Carey et al, 2008, J Clin
Path)
• 100% PA w/ 1:10 saline dilution of blood culture broth
(Varrettas, et al, 2005, J. Clin Microbiol)
• False Negative = 1 - % Positive Agreement (PA)
• False Positive Risk is Low
• S. intermedius and S. hyicus (animal strains) are positive by the
tube test (generally a delayed reaction)
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Oxoid PBP2’ Latex Agglutination
• N = 25, small sample size
• Heaping loop of growth from film
on subculture
• Results emailed to ID Pharmacist
• 100 % agreement with oxacillin
MIC
• 17% of positives, Rx switched due
to rapid test, either broaden or
narrow the Rx coverage
Marlowe, 2002. et al, Microbiol. Infect. Dis.
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Direct Thermonuclease Test
• Thermonuclease agar
• Toluidine blue dye: blue when complexed
with DNA; Pink zone when DNA breaks
down to nucleotides
• Accurate when compared to DTCT
Kaplan, 2003 Eur Med Health Journ
• Some CoNS can destroy dye w/out
denaturing DNA
• No outcome studies
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Direct Pneumoslide
• BD BBL™ Pneumoslide™ Test for
Streptococcus pneumoniae
• 32/32 from positive broth aliquot
• No outcome studies
•
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Davis et al, 1992, DMID
Examples, Direct Vitek and Phoenix
Two Recent Publications
• 95.8% correct ID n = 136/142
• 2.8% error for Abx
• Munoz-Davila, 2011 Eur J. Clin Micro Inf
% Agreement
w/ Ref Std
Gram Neg
Gram Pos
Vitek 2
100
75
BD Phoenix
92.3
43.7
Gherardi et al, 2012 JCM
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Impact Studies Vitek 1 or 2 (low n)
• Decreased Abx dose/patient by 6 d, p = 0.02
Kerremans et al, 2008, JAAC
• Direct Testing reduced report time, 48 hr to 8.8 hr
• Direct method significantly more likely to result in
change to Rx than routine method
Trenholme et al, 1989, J Clin Microbiol
• No change in impact variables, with dayshift only
Bruins et al, 2005, Eur J Clin Microbiol Inf Dis
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Molecular Methods
PNA FISH
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Fluorescence from Probe bound to rRNA in Bacteria
Fix Slide, Hybridize, Wash/Mount, Read w/ Fluorescent Scope
97-100% sensitivity and specificity per insert
BacT/Alert, Bactec, or TREK
E. faecalis (green)
Non-faecalis enterococci (red)
T.A.T.= 1.5 Hr
H.O.T. = 12min.
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Enterococcus spp., Reduction in Mortality/Costs
E. faecalis/OE PNA FISH
• E. faecium
– Decreased 30 d
mortality from 45%
to 26%, p = 0.04
– Savings $20,000/yr
Forrest, et. al 2008, Antimicrob
Agents Chemother
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Staphylococcus spp., Reduction Mortality/Costs/LOS
S. aureus/CNS PNA FISH®
• Staphylococcus spp.
– 80% reduction in ICUrelated mortality
– 53% overall reduction
– Median saved $19,441/
patient
Ly et, al, 2008. Ther Clin Risk
Manag. 4(3):637-40
– Vancomycin days
reduced, 4.8 to 2.5 d,
p=0.06
– LOS decreased from 6 to
4d/case, p < 0.05
Forrest et al JAAC, 2006
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Early indications – requires lab and
pharmacy to work with clinicians for
intervention (Holtzman, C. et al, Jan 2011,
J Clin Microbiol)
Candida: Reduction in Mortality/Costs
C. albicans/C. glabrata PNA FISH®
• C. albicans
• Decreased 30 d mortality
(26% vs. 45%; p = 0.04)
• Decreased capsofungin
usage from 8.7 to 3.2 d
• Savings $61,348/patient
Forrest, G. N. et. al 2006, J Clin
Microbiol
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UAMC Experience: E. faecalis /OE PNA FISH®
GPC Pairs and Chains, 262 PNA FISH of Total n = 460
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Gamage et al, 2011 ICAAC Abstract D-1302b
UAMC Experience: Candida spp.
C. albicans/C. glabrata PNA FISH®
82 PNA FISH of Total n= 125
Gamage et al, 2011 ICAAC Abstract D-1302b
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Others Reports of Shortened Length of Stay
PNA FISH vs. Conventional Methods
33 7 days
26 11 1. Candida
(All)
2 days
9 2. Staphylococci
(All)
Control Group
1)
2)
3)
4)
30
9 2 days
7 3. CoNS
(All)
PNA FISH
Virgina Mason Medical Center: Poster C 2563. ASM 2011. New Orleans, Louisiana, USA.
Orlando Regional Medical Center: Poster 1023. IDSA 2010. San Diego, California, USA.
Washington Hospital Center: Ther Clin Risk Manag. 2008 Jun;4(3):637-40.
University of Maryland Medical Center: J Antimicrob Chemother. 2006 Jul; 58(1): 154-8.
6 2 days
4 4. CoNS
(Contamination)
Molecular Methods
Real-time PCR
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GeneXpert® MRSA/SA detection
• Avoids false + from vestigial SCCmec cassette, n=406
–
–
–
–
MRSA: 98.3% pos. agreement; 99.4% negative agreement
MRSA LOD = UCL 128 CFU/Sample*
SA 100% pos agreement, 98.6% neg. agreement
SA LOD = UCL 57 CFU/Sample*
• FYI: BC Flags pos. at ~ 1.2 × 108 (Marlowe et al, JCM
2003); Subculture still ? more sensitive @ < 30 -3,000/mL
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Deliver + answers from
sample in < 50 min.
*Wolk, et. al, 2009. J Clin Microbiol
NOTE:
LDT if
charcoal
or resin
bottles
GeneXpert® MSSA Bacteremia Intervention
Decreased Vancomycin Use, LOS, Costs
• Multivariable regression (n=156)
– Mean de-escalation time: empiric vancomycin 
cefazolin/nafcillin = 1.7 days shorter (p = 0.002)
– Mean LOS = 6.2 days shorter (p = 0.07)
– Mean hospital costs = $21,387 less (p = 0.02)
Bauer et al. CID 2010
33
Some LDTs, PCR for S. aureus
• Nguyen et al, 2010
• Vancomycin therapy was de-escalated more often to
targeted therapy after intervention 73.4% vs. 47.7%
• Stellrecht et al, 2009, ICAAC
• Median vancomycin/MSSA infxn. reduced, 4 to 2 g
• Hallin et al, JCM, 2003
• 25% benefited from a modification of Abx therapy
based on the PCR (low n)
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BD GeneOhm StaphSR assay
Accuracy study
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• Grobner et al, 2009, JCM
– No outcome studies
– Detection of S. aureus and MRSA
– MSSA (n = 90); MRSA (n = 45)
– MSSA sensitivity/specificity: 95% CI’s (96.0 to 100% /
82.4 to 100%)
– MRSA 95.6% sensitive (95% CI, 84.9 to 99.5%),
95.3% specific (95% CI, 86.9 to 99.0%)
– 5 discrepant results
• presence of methicillin-susceptible, revertant
MRSA strains (3/45)
• MRSA strains that were not detected (2/45)
QuickFISH Technology
• Proprietary PNA probe/quencher technology
• Unique probe/quencher complexes allow for:
– Fluorescence signal in the presence of target rRNA
– Fast hybridization; No Wash; 20min, < 5min H.O.T.
Fluorophore
Deck, MK et al, J Clin Microbiol,
eprint 4/4/2012
PNA Probe
Sequence
S. aureus Sensitivity very good
Specificity ok
Quencher
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QuickFISH – One Decision Point
Sepsis: Empiric Rx
Rx Change
Blood Cultures
Critical Values Call
16-24 Hrs
Microbiology
37
Gram stain
Pathogen ID
(Positive BC)
(QuickFISH)
BioFire FilmArray BCID
38
FilmArray BCID
39
FilmArray BCID
40
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•
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•
•
•
•
•
•
Gram Positive Bacteria
Staphylococcus spp.
Staphylococcus aureus
Enterococcus spp.
Streptococcus pneumoniae
Streptococcus spp.
Streptococcus agalactiae
Streptococcus pyogenes
Listeria monocytogenes
•
•
•
•
•
•
Fungi
Candida albicans
Candida krusei
Candida glabrata
Candida parapsilosis
Candida tropicalis
•
•
•
Gram Negative Bacteria
Acinetobacter baumanii
Enterobacteriaceae
–
–
–
–
–
–
•
•
•
•
Enterobacter cloacae
Escherichia coli
Klebsiella oxytoca
Klebsiella pneumoniae
Proteus spp.
Serratia marcescens
Haemophilus influenzae
Haemophilus influenzae?typeable
Neisseria meningitidis
Pseudomonas aeruginosa
Antibiotic Resistance Genes
• mecA (methicillin resistance)
• vanA/B (vancomycin resistance)
• blaKPC (carbapenam resistance)
Nanosphere
BC-GP Assay
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Species
US/FDA-Cleared
BC-GP assay
Staphylococcus aureus
x
Staphylococcus epidermidis
x
Staphylococcus lugdunensis
x
Streptococcus anginosus Group
x
Streptococcus agalactiae
x
Streptococcus pneumoniae
x
Streptococcus pyogenes
x
Enterococcus faecalis
x
Enterococcus faecium
x
Genus
Staphylococcus spp.
x
Streptococcus spp.
x
Micrococcus spp.
Listeria spp.
x
Resistance
42
mecA
x
vanA
x
vanB
x
Nanosphere
Verigene® Reader and Processor SP
Nanosphere, BC-GP Assay
• Linoj et al, JCM 2013. 51(4) 1188-1192
– N= 203 VersaTrek bottles
– 92% agreement for identification
– 96% agreement for resistance
– 6/25 polymicrobial infections not detected
• Wojewoda et al, 2013 JCM Apr 17
– Agreement 94.6%
– 40% correct for S. pneumoniae
– Van A good
– 3 mec results from non S. aureus non S. epi
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Other Technologies of Interest
BC Bottles and Whole Blood
44
MicroPhage KeyPath™
MRSA/MSSA Blood Culture
Test
45
MicroPhage KeyPath™ MRSA/MSSA
Blood Culture Test
FDA cleared, 5 hours
46
mecA PNA FISH
• ECCMID 2012, Preliminary Data
• mRNA target
• Bactec™ and BacT/Alert™ bottles, 2 hr TAT
• 100% for MRSA for n=18
• 100% for MSSA n=72
• Missed MRCoNS (delayed expression?)
47
Matrix Assisted Laser
Desorption Ionization (MALDI)
Bruker Daltonics MALDI BioTyper (TM)
bioMérieux = Vitek MS
Measure and compare high abundance proteins
48
Bruker SepsiTyper
Hopkins, Brown, UA
ASM Poster, 2012
49
UA/JHH/RI Hospital Study
Sepsityper 381 samples
JHH
n=226, Bactec
Brown
n=155, TREK
34 mixed organism samples excluded
------------------------------------------------------------347 single-organism samples
(45 species by conventional methods)
230 gram-positive bacteria
103 gram-negative bacteria
14 yeasts
50
Median time from BC + to extraction was 4.3 h
Results UA/JHH/RI, n=347
n
Agreed with Conventional
Discordant
51
275
9
No Reliable ID
16
Insufficient protein
47
Vlek et al, PLoS One, 2012
• Direct MALDI-TOF Improves Appropriateness of
Antibiotic Treatment of Bacteremia
• 11.3% increase in the patients w/ appropriate Rx
24 hours after blood culture positive
• (75.3% vs 64.0% (p = 0.01).
52
Strategy for Rapid
ID/Susceptibility
• Tested Gram-Negative Bacteria from Positive
Blood Cultures
– Bruker MALDI Biotyper coupled w/ rapid
susceptibility testing (BD Phoenix)
• Wimmer, et al, JCM 2012
53
MRSASelect and SASelect
(BioRad)
for Staphylococci
Schweizer et al. Comparative effectiveness of
nafcillin or cefazolin versus vancomycin in
methicillin-susceptible Staphylococcus aureus
bacteremia. BMC Infectious Diseases 2011
11:279.
54
MRSASelect and SASelect agar.
• UA 484 BC, off-label practice of 12 hour identification
• Chromogenic agars examined after 12 and 24 hours of
incubation at 35°C in non-CO2
• Equivalent performance vs. traditional ID
– Sensitivity 99.8%, specificity 100%, agreement 99.8%
• Prevalence of SA was 18.5% (47.4% of these MRSA)
(overall prevalence15.7% MRSA, 22.1% MSSA)
• The LOD for MRSASelect was 5 cfu/mL
55
• One false negative with a treated MRSA
Other Technologies of Interest
BC Bottles
• Pyrosequencing
Jordan et al, 2005, J Clin Microbiol
Jordan et al, 2009, J Clin Microbiol
Quiles-Melero et al, 2011, Eur J. Clin Micro Inf
• PCR electrospray Ionization Mass Spectrometry
(PCR/ESI-MS)
Kaleta, E.J., et al . 2011. J. Clin. Microbiol., 49(1), 345-353.
Kaleta, E.J., Clark, A.E., et al, 2011 Clin. Chem. 57(7), 1-11
56
Tier Two
Emerging Technologies
Directly from Whole Blood
No interventional studies to date
57
Directly from Whole Blood?
•
•
•
•
Roche LightCyler® SeptiFast
Molzyme Universal Microbe Detection
Next Generation Sequencing ???
Ion Torrent Sequencing ???
• Extraction Technologies will be key
o 10ml extraction (Molzyme Qiagen, ABI, etc)
58
NEW: From whole blood
in ~ 3 hours
T2 Bacteria
T2 Candida
59
The T2 Magnetic Resonance (T2MR®)
• Measure dynamics of water
• Direct detection of biomarkers in complex
samples such as whole blood, plasma,
serum, sputum and urine
• Wide range of analyte types, including
pathogens, genomics, proteins, smallmolecule immunochemistry and
hemostasis measurements.
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60
BACcel™ Diagnostic Platform
Next generation antimicrobial
susceptibility testing methods
61
Track Individual Clones
Acinetobacter
2 divisions
Computer-imposed
cell mass contours
(color ellipsoids)
Pseudomonas
2 divisions
62
Summary Highlights
• BSIs challenge clinical laboratories to provide integrated care
• More evidence-based studies are needed to assess cost-benefit of
rapid testing
• Early indications – requires lab and pharmacy to work with clinicians
for intervention (Holtzman, C. et al, Jan 2011, J. Clin. Microbiol.)
• Take care with implementation of emerging technology
– More development of the databases is needed
– Standardization to obtain comparable results
– Develop practice guidelines/algorithms for clinicians/pharmacists
63
The Future
CDC/ASM Laboratory
Medicine Best Practices
(LMBP)
https://www.futurelabmedicine.org/
64
Acknowledgements
65
•
IDRC: Desiree Johnson, Andrew Clark,
Dulini Gamage, Joseph Marano, Elizabeth
Ingram, Erica Isaacs, Sharon Hooven,
Daniel Olson
•
Wysocki Research Group: Dr. Vicki
Wysocki, Erin Johnson (NIH BCP Training
Grant)
•
UMC: Wanda Petty, Lorraine Franco, Ellen
Tuttle, Laurel Burnham, Bruce Anderson
Katie Mathias
•
University of Geneva Hospital: Jacques
Schrenzel, Abdessalam Cherkaoui
Questions?
Questions
dmwolk@geisinger.edu
Advancing diagnostics….saving lives
Heal • Teach • Discover • Serve
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