Blood Culture Nucleic Acid Testing: Identification and Resistance
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Transcript of Blood Culture Nucleic Acid Testing: Identification and Resistance
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The UC San Diego AntiViral Research Center sponsors weekly presentations by infectious disease clinicians, physicians and researchers. The goal of these presentations is to provide the most current research, clinical practices and trends in HIV, HBV, HCV, TB and other infectious diseases of global significance. The slides from the AIDS Clinical Rounds presentation that you are about to view are intended for the educational purposes of our audience. They may not be used for other purposes without the presenter’s express permission.
AIDS CLINICAL ROUNDS
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Blood Culture Nucleic Acid Testing: Bacterial Identification
and Resistance
David Ha, PharmD PGY2 Pharmacy Resident, Infectious Diseases
UCSD Health System AIDS Rounds
June 20, 2014
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Disclosures
• None
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Blood Culture Rapid Diagnostics
• Conventional bacterial identification and susceptibility testing methods take, on average, 3-5 days to finalize
• Molecular methods (e.g. PCR, PNA-FISH or direct NAT) may reduce time to appropriate therapy, infection-related morbidity/mortality and cost
• Molecular methods utilize detection of trace amounts of bacterial DNA in a blood culture sample to identify bacteria genera and species and detect presence of resistance mechanisms
Wojewoda et al. J Clin Microbiol 2013; 51(7):2072-6
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Blood Culture Nucleic Acid Tests
• NAT (direct nucleic acid testing)
– Nanosphere Verigene BC-GP (GP), BC-GN (GN)
• PCR (polymerase chain reaction)
– Biofire FilmArray BCID (GP/GN/yeast) – BD GeneOhn StaphSR (MRSA) – Cepheid GeneXpert MRSA/SA BC (MRSA)
• PNA-FISH (peptide assisted nucleic acid - fluorescence in-situ hybridization)
– AdvanDx (GP/GN/yeast)
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Verigene® Blood Culture Nucleic Acid Test
• Qualitative multiplexed nucleic acid assay (no amplification)
• Performed directly on whole blood
• Detect bacterial DNA (identification)
• Detect resistance genes (resistance)
• Sample Result ~1.5 hr
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Test Principle
• 4 General Phases – Cell Lysis – Extraction
• Magnetic bead-based DNA extraction – Hybridization
• Bacterial DNA hybridizes to capture DNA on gold nanoparticles in a microarray format
– Identification • Silver enhancement of microarray creates silver-gold
aggregates that are optically imaged
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Test Principle
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Methodology
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Methodology
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Methodology
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Methodology
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Applicability to Antibiotic Stewardship
Detection of Bacterial DNA and
Resistance Mechanisms
MD or IDPharmD
notified
Therapy Evaluated and Changed if
Necessary
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Blood Culture Gram Positive Test (BC-GP)
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BC-GP Intended Use
Bacterial Species Bacterial Genera Resistance
Staphyloccocus aureus Staphylococcus epidermidis Staphylococcus lugdunensis
Streptococcus anginosus group Streptococcus agalactiae
Streptococcus pneumoniae Streptococcus pyogenes
Enterococcus faecalis Enterococcus faecium
Staphylococcus spp.
Streptococcus spp.
Listeria spp.
mecA
vanA
vanB
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BC-GP Identification Performance Bacterial Species n PPA NPA
S. aureus 1,426 99.1% 100%
S. epidermidis 1,426 93.1% 100%
S. lugdunensis 1,426 95.0% 100%
S. anginosus group 1,426 100% 99.8%
S. agalactiae (GBS) 1,426 98.6% 100%
S. pneumoniae 1,426 100% 99.6%
S. pyogenes (GAS) 1,426 95.8% 100%
E. faecalis 1,426 96.9% 99.9%
E. faecium 1,426 97.1% 100%
Bacterial Genera n PPA NPA Staphylococcus spp. 1,426 98.0% 99.4%
Streptococcus spp. 1,426 93.6% 99.6%
Listeria spp. 1,426 100% 100%
Reference Method: Culture and Conventional Biochemical and Phenotypic Identification
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BC-GP Intended Use
Bacterial Species Bacterial Genera Resistance
Staphyloccocus aureus Staphylococcus epidermidis Staphylococcus lugdunensis
Streptococcus anginosus group Streptococcus agalactiae
Streptococcus pneumoniae Streptococcus pyogenes
Enterococcus faecalis Enterococcus faecium
Staphylococcus spp.
Streptococcus spp.
Listeria spp.
mecA
vanA
vanB
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mecA-encoded Methicillin Resistance
M
MSSA mecA-
Methicillin-Sensitive
Methicillin
mecA
ON
MRSA mecA+
Methicillin-Resistant
PBP-2a
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vanA/vanB-encoded Vancomycin Resistance
Source: http://dc245.4shared.com/doc/h4Xa1-sL/preview.html. Accessed: 12/20/2013
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BC-GP Resistance Performance
Bacterial Species n PPA NPA
S. aureus 335 97.5% (157/161)
98.8% (172/174)
S. epidermidis 330 92.0% (219/238)
81.5% (75/92)
E. faecalis 109 85.7% (12/14)
100% (95/95)
E. faecium 114 97.2% (69/71)
93.0% (40/43)
E. faecalis 109 100% (7/7)
100% (102/102)
E. faecium 114 97.0% (32/33)
100% (81/81)
mecA
vanA
vanB
Methicillin Resistance
Vancomycin Resistance
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BC-GP Performance
• Excellent identification performance of all species and genera
• Excellent resistance performance for methicillin and vancomycin resistance – Slightly lower specificity with mecA in S.
epidermidis
• Time to result: ~1.5 hours
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BC-GP Clinical Utility
• Organism Identification – Source
• Escalation and De-escalation of therapy – Differentiate methicillin-resistant vs. methicillin-
sensitive S. aureus and S. epidermidis – Differentiate vancomycin-resistant vs. vancomycin-
sensitive E. faecalis and E. faecium • Negative Results (less utility)
– Absence of detectable organism is not clinically useful as it could imply non-target bacteria
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Blood Culture Gram Negative Test (BC-GN)
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Intended Use
Bacterial Genera and Species Resistance Markers
Acinetobacter spp. Citrobacter spp.
Enterobacter spp. Proteus spp.
Escherichia coli* Klebsiella pneumoniae
Klebsiella oxytoca Pseudomonas aeruginosa
CTX-M KPC
NDM VIM IMP OXA
* Cannot be distinguished from Shigella spp. (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei)
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Organism Isolates (N) PPA NPA
Acinetobacter spp. 1412 98.2% (55/56)
99.9% (1355/1356)
Citrobacter spp. 1412 100% (49/49)
99.9% (1362/1363)
Enterobacter spp. 1412 97.6% (120/123)
99.4% (1281/1289)
Proteus spp. 1412 100% (58/58)
99.9% (1353/1354)
Escherichia coli 1412 99.8% (517/518)
99.4% (889/894)
Klebsiella pneumoniae 1412 93.1% (285/306)
100% (1106/1106)
Klebsiella oxytoca 1412 92.2% (59/64)
99.6% (1342/1348)
Pseudomonas aeruginosa 1412 97.6% (124/127)
100% (1285/1285)
Reference Method: Culture and Conventional Biochemical and Phenotypic Identification
BC-GN Identification Performance
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Intended Use
Bacterial Genera and Species Resistance Markers
Acinetobacter spp. Citrobacter spp.
Enterobacter spp. Proteus spp.
Escherichia coli* Klebsiella pneumoniae
Klebsiella oxytoca Pseudomonas aeruginosa
CTX-M KPC
NDM VIM IMP OXA
* Cannot be distinguished from Shigella spp. (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei)
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Beta-Lactamases
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Classification of Beta-Lactamases
• Molecular – Ambler Classification System
• Functional – Bush-Jacoby-Medeiros Grouping System
• Clinical – “ESBL”, “Cephalosporinase”, “Carbapenemase”
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Classification of Beta-Lactamases
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Beta Lactamases Detected by BC-GN Panel
Target Enzyme Potential Spectrum Phenotypic Group
CTX-M Cefotaxime-resistant beta lactamase Penicillins, Cephalosporins ESBL
KPC Klebsiella pneumoniae carbapenemase
Penicillins, Cephalosporins, Carbapenems Carbapenemase
NDM New Delhi Metallo-beta lactamase
Penicillins, Cephalosporins, Carbapenems Carbapenemase
VIM Verona Integron-encoded Metallo-beta lactamase
Penicillins, Cephalosporins, Carbapenems Carbapenemase
IMP Imipenem-Resistant Metallo-beta lactamase
Penicillins, Cephalosporins, Carbapenems Carbapenemase
OXA Oxacillinase Penicillins, +/- Cephalosporins, Carbapenems
Carbapenemase (+/-
Cephalosporinase)
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The Clinical Conundrum
Genetic Resistance
Resistance by AST
Clinical Failure
? ?
Absence of Genetic
Resistance
Susceptibility by AST
Clinical Efficacy
? ?
Molecular Testing
Conventional Susceptibility
Testing
Clinical Result
? ?
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PPA = TP / (TP + FN) NPA = TN / (TN + FP)
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CTX-M…the ESBL Phenotype • PPA < 40% for Acinetobacter spp., Citrobacter spp.,
Enterobacter spp., K. pneumoniae, K. oxytoca, P. aeruginosa – False negatives are common – If no CTX-M detected, ESBL-like resistance is still possible – Other beta lactamases (blaAmpC, blaTEM, blaSHV) and non-beta
lactamase resistance mechanisms not assayed for • NPA > 90% for all isolates
– False positives are uncommon – If CTX-M detected, high likelihood of ESBL-like resistance
• Escherichia coli
– PPA ~ 82% = False negatives appear to be uncommon – If no CTX-M detected, somewhat low likelihood of ESBL resistance – Alternative ESBLs less common in E. coli than in other genera
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PPA = TP / (TP + FN) NPA = TN / (TN + FP)
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Carbapenem-Resistance • PPA ≥ 90% for Acinetobacter spp., Citrobacter spp.,
Enterobacter spp., E. coli, K. pneumoniae, K. oxytoca – False negatives are uncommon – If no resistance detected, low likelihood of carbapenem
resistance (if not Pseudomonas aeruginosa) • NPA ≥ 90% for ALL isolates
– False positives are uncommon – If resistance is detected, high likelihood of carbapenem
resistance
• Pseudomonas aeruginosa – PPA = 12.5% = False negatives common – If no resistance detected, carbapenem resistance still possible – Altenative resistance mechanisms to carbapenems (i.e. loss of
OprD porin, efflux pumps, etc)
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BC-GN Performance • Excellent identification performance of all species and
genera • CTX-M for ESBL phenotype
– CTX-M Present – Use Carbapenem – CTX-M Absent – Not clinically useful (good chance E. coli is
susceptible to 3rd gen cephs) • KPC, NDM, VIM, IMP, OXA for carbapenemase
phenotype – Present – Use non-beta-lactam – Absent – Can use Carbapenem (P. aeruginosa may still be
resistant) • Time to result: ~1.5 hours
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BC-GN Clinical Utility
• Organism Identification – Source
• Mainly escalation of therapy – Anti-pseudomonal antibiotics when Pseudomonas
aeruginosa detected – Carbapenems for CTX-M positive bacteria – Non-beta lactams for KPC, NDM, VIM, IMP, OXA
positive bacteria • Potentially de-escalation
– Appropriate non-antipseudomonal antibiotics when non-P. aeruginosa organism detected and no polymicrobial infection suspected
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BC-GN Clinical Utility
• Negative Results (less clinical utility) – Absence of detectable organism is not clinically
useful as it could imply non-target bacteria – Absence of CTX-M does not predict non-ESBL
phenotype (except perhaps in E. coli) – Absence of KPC, NDM, VIM, IMP, OXA predicts
carbapenem susceptibility well in all isolates except P. aeruginosa
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BC-GP/BC-GN Test Limitations
• Subpopulations – Mixed culture/resistance – Low prevalence of resistance marker
• Sample Specificity – BACTEC™, BacT/ALERT®, VersaTREK® REDOX
• Target Specificity – Sequence variants in target bacteria – Non-target bacterial homology – Non-target resistance mechanisms (blaAmpC, blaTEM,
blaSHV) • In vitro vs. in vivo Resistance
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BONUS! MALDI-TOF MS
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Matrix Assisted Laser Desorption Ionization Time Of Flight Mass Spectrometry
What is MALDI-TOF MS?
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Direct Smear Method: 1. Touch colony with transfer device, such as toothpick 2. Transfer a small amount onto spot, let air dry (+/- FA, air dry) 3. Cover with 1 µL of MALDI matrix, let air dry 4. Load target into machine Analyze
Sample/target preparation for most bacterial isolates
Research use only – not for use in diagnostic procedures
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Target plate
Analyte (organism)
1 µL Matrix
Matrix Assisted Laser Desorption/Ionization
Matrix: HCCA (α-Cyano-4-hydroxycinnamic acid) Solvent: Acetonitrile, TFA (trifluoroacetic acid)
• Lyses cell walls and extract protein • Separates protein molecules (proteins are “sticky”)
Research use only – not for use in diagnostic procedures
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Matrix Assisted Laser Desorption/Ionization
• Laser light pulses
• Matrix molecules readily absorb laser light (photon energy), creating an excited energy state
• The matrix is acidic, and donates positive charge to the analytes
Research use only – not for use in diagnostic procedures
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Matrix Assisted Laser Desorption/Ionization
Matrix
• Localized heating causes micro-explosion of material
• Collisions with neutral sample facilitate charge transfer to/from excited matrix molecules
• Ions “desorb” from the target surface
Research use only – not for use in diagnostic procedures
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Drift region
Detector
TOF – Time of Flight
m/z
Intensity
• Following acceleration, the charged ions are allowed to drift through a free field toward the detector
• The speed of travel (time of flight) is proportional to the ion’s mass (smaller ions reach the detector first)
Research use only – not for use in diagnostic procedures
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How reliable are identifications?
Croxatto et al. 2011
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Special Thanks
• Michele Jasura, MT/CLS • Sanjay Mehta, MD • Heather Fritz, DVM • Sharon Reed, MD • David Pride, MD • Charles James, PharmD
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Additional Slides
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BC-GN Genetic Targets Genetic Target Bacterial/Resistance Correlate
rpsA Acinetobacter spp.
ompA/mrkC Citrobacter spp.
gyrB/metB Enterobacter spp.
atpD Proteus spp.
oppA Escherichia coli
yggE Klebsiella pneumoniae
ompA Klebsiella oxytoca
sodA Pseudomonas aeruginosa
blaCTX-M CTX-M
blaKPC KPC
blaNDM NDM
blaVIM VIM
blaIMP IMP
blaOXA OXA
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BC-GN Controls
• Internal – Fluid control, hybridization, signal enhancement – INT CTL 1 = Artificial DNA oligonucleotide – INT CTL 2 = Shewanella oneidensis DNA
• External – Initial test validation – Ongoing QC validation
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BC-GN Potential False Results Result FP/FN Potential Interpretation
Escherichia coli FP Shigella spp. including S. dysenteriae, S. flexneri,
S. boydii, and S. sonnei. FP Escherichia albertii
Citrobacter spp. FP Buttiauxella gaviniae and Enteric group 137 (ATCC BAA-69)
Klebsiella oxytoca FP Kluyvera ascorbata, Raoultella ornithinolytica, Raoultella planticola, and Cedecea davisae
CTX-M FP Leminorella grimontii, Enterococcus raffinosus and Candida parapsilosis
Acinetobacter spp. not detected FN Acinetobacter tartarogenes
Enterobacter spp. not detected FN Enterobacter gergoviae, Enterobacter kobei, and
Enterobacter pyrinus
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Alternative Applications (Off-Label)
• Direct Specimen – Suspend bacterial colonies in saline and add to
sample cartridge
• Non-Blood Specimen – CSF