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: 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.
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
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
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