Blood Culture Nucleic Acid Testing:  Identification and Resistance
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Blood Culture Nucleic Acid Testing: Identification and Resistance

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David Ha, PharmD, of UC San Diego Health System, presents "Blood Culture Nucleic Acid Testing: Identification and Resistance" at AIDS Clinical Rounds

David Ha, PharmD, of UC San Diego Health System, presents "Blood Culture Nucleic Acid Testing: Identification and Resistance" at AIDS Clinical Rounds

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    Blood Culture Nucleic Acid Testing:  Identification and Resistance Blood Culture Nucleic Acid Testing: Identification and Resistance Presentation Transcript

    • 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
    • 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
    • Disclosures • None
    • 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
    • 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)
    • 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
    • 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
    • Test Principle
    • Methodology
    • Methodology
    • Methodology
    • Methodology
    • Applicability to Antibiotic Stewardship Detection of Bacterial DNA and Resistance Mechanisms MD or IDPharmD notified Therapy Evaluated and Changed if Necessary
    • Blood Culture Gram Positive Test (BC-GP)
    • 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
    • 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
    • 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
    • mecA-encoded Methicillin Resistance M MSSA mecA- Methicillin-Sensitive Methicillin mecA ON MRSA mecA+ Methicillin-Resistant PBP-2a
    • vanA/vanB-encoded Vancomycin Resistance Source: http://dc245.4shared.com/doc/h4Xa1-sL/preview.html. Accessed: 12/20/2013
    • 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
    • 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
    • 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
    • Blood Culture Gram Negative Test (BC-GN)
    • 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)
    • 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
    • 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)
    • Beta-Lactamases
    • Classification of Beta-Lactamases • Molecular – Ambler Classification System • Functional – Bush-Jacoby-Medeiros Grouping System • Clinical – “ESBL”, “Cephalosporinase”, “Carbapenemase”
    • Classification of Beta-Lactamases
    • 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)
    • 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 ? ?
    • PPA = TP / (TP + FN) NPA = TN / (TN + FP)
    • 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
    • PPA = TP / (TP + FN) NPA = TN / (TN + FP)
    • 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)
    • 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
    • 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
    • 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
    • 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
    • BONUS! MALDI-TOF MS
    • Matrix Assisted Laser Desorption Ionization Time Of Flight Mass Spectrometry What is MALDI-TOF MS?
    • 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
    • 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
    • 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
    • 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
    • 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
    • How reliable are identifications? Croxatto et al. 2011
    • Special Thanks • Michele Jasura, MT/CLS • Sanjay Mehta, MD • Heather Fritz, DVM • Sharon Reed, MD • David Pride, MD • Charles James, PharmD
    • Additional Slides
    • 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
    • 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
    • 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
    • Alternative Applications (Off-Label) • Direct Specimen – Suspend bacterial colonies in saline and add to sample cartridge • Non-Blood Specimen – CSF