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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