RAPID IDENTIFICATION OF RESISTANCE MECHANISMS
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1 RAPID IDENTIFICATION OF RESISTANCE MECHANISMS Christine C. Ginocchio, PhD, MT (ASCP) Professor of Medicine, Hofstra North Shore-LIJ School of Medicine, NY VP, Global Microbiology Affairs, biomerieux VP, Global Medical and Scientific Affairs, BioFire Antimicrobials 2015, Brisbane, Australia
2 Clinical History: Jane Doe A 58 year old female with a history of COPD, multiple past hospital admissions collapses at home and it brought by ambulance to the Emergency Department Patient in distress, temperature o F, confused, short of breath, tachycardic, hypotensive, productive cough, chest pain Preliminary Tests: WBC: elevated at 18,000 Lactate is elevated Urine urinalysis is normal, culture ordered Sputum culture ordered Chest radiograph is normal, no infiltrates, consolidations etc Transferred to ICU 2
3 Duration of Hypotension and Initiation of Appropriate Antibiotic Therapy ED PCT: 3.25 PCT: 9.75 PCT: 16.5 UC-/BC+ Growth ID/AST Etest PCT: 16.0 UC/BC/AbX Lab Hours Kumar A, et al.: Crit Care Med, 34: ,
4 Resistance Detection from Direct Culture
5 Primary Screening: Chromogenic Agars MRSA, VRE, ESBL, KPC, CRE Performance varies Save hr 5
6 The time to targeted therapy was significantly lower (P=0.006) in Case patients (mean; h) versus Control patients: (mean; h) Rapid testing yielded a significant result, with a difference of 12.2 h to targeted therapy Time to susceptibility test result between the full traditional methodology and CHROMagar was even larger (26.5 h) 6
7 Detection of meca Mediated Resistance Latex agglutination sensitivity meca detection: 92.3% to 97.29% In comparison to direct PCR 8
8 Rapid Detection of Carbapenemaseproducing Enterobacteriaece 9
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11 3 hr for Gram Neg 4 hr for Gram positive 12
12 SCMA results in <4 hr. 91.5% categorical agreement, 6.51% minor, 2.56% major, 1.48% very major errors 13
13 Resistance Detection from Blood Cultures
14 AdvanDx PNA FISH Technology Fluorescence tagged peptide nucleic acid probes detects 16S rrna directly from positive blood cultures (G): S. aureus (G): S. aureus (R): CoNS (G) E. faecalis (R) other Enterococcus spp. (G): C. albicans (G): C. albicans (R): C. glabrata (G): C. albicans/c. parapsilosis (R): C. glabrata/c. krusei (Y): C. tropicalis 15
15 Assuming physician notification: potential savings was $1,837/patient PNA FISH: $82.72, C. albicans screen: $
16 18
17 Accelerate Diagnostics Sample undergoes simple cleanup procedure, is added to the cassette which is inserted into the Accelerate ID/AST system. Any sample type can be used. The system extracts, immobilizes, and maps bacteria in sample. Microscope image: specks are individual bacteria, concentrated directly from a sample and immobilized for analysis. Automated susceptibility testing: After starting antibiotic exposure, susceptible cells begin to die (red stain) and resistant cells keep on growing (green). Yellow indicates injured cells that will soon die. The Accelerate ID/AST system reports susceptibility interpretation and resistance expression. Time: 2-4 hrs 19
18 An aliquot of the blood culture was subcultured in the presence of specific antibiotics and bacterial counts were monitored using the Sysmex UF-1000i flowcytometer at different times up to 180 min. Receiver operating curve (ROC) analysis allowed us to find out the cut-off point for differentiating between sensitive and resistant strains to the tested antibiotic. AST can be determined as early as 120 min since a blood culture bottle is flagged as positive. Essential agreement between our susceptibility test and commercial methods (E-test, MicroScan and Vitek) was 99%. 20
19 Detection of MSSA/MRSA from Positive Blood Broths BD-GeneOhm StaphSR Collected Specimen Specimen Preparation Lysis - DNA Extraction Reconstitution Of Reagents Results within 2 Hours Real-time PCR Analysis on the SmartCycler Definitive On-screen Results GeneXpert MRSA/SA-Cepheid 1 hour detection meca specific and detect chromosomal deletion isolates as meca positive 21
20 ID PharmaD was contacted with results of the rpcr; (Xpert MRSA, Cepheid) effective antibiotics and an infectious diseases consult were recommended. Multivariable regression assessed clinical and economic outcomes of the 156 patients. Mean time to switch from empiric vancomycin to cefazolin or nafcillin in patients with methicillinsusceptible S. aureus bacteremia was 1.7 days shorter post-rpcr (P=0.002). In the post-rpcr methicillin-susceptible and methicillinresistant S. aureus groups, the mean length of stay was 6.2 days shorter (P=0.07) and the mean hospital costs were $21,387 less (P=0.02). rpcr allows rapid differentiation of S. aureus bacteremia, enabling timely, effective therapy and is associated with decreased length of stay and health care costs. 22
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22 Nanosphere Verigene Blood Culture ID Panels Gram Positive Targets US/FDA-Cleared Outside US Species Staphylococcus aureus X X Staphylococcus epidermidis X X Staphylococcus lugdunensis X X Streptococcus anginosus Group X Streptococcus agalactiae X X Streptococcus pneumoniae X X Streptococcus pyogenes X X Enterococcus faecalis X X Enterococcus faecium X X Genus Staphylococcus spp. X X Streptococcus spp. X X Micrococcus spp. X Listeria spp. X X Resistance meca (methicillin) X X vana (vancomycin) X X vanb (vancomycin) X X X Gram Negative Targets US/FDA-Cleared Outside US Species Escherichia coli 1 X X Klebsiella pneumoniae X X Klebsiella oxytoca X X Pseudomonas aeruginosa X X Serratia marcescens X Genus Acinetobacter spp. X X Citrobacter spp. X X Enterobacter spp. X X Proteus spp. X X Resistance CTX-M (ESBL) X X IMP (carbapenemase) X X KPC (carbapenemase) X X NDM (carbapenemase) X X OXA (carbapenemase) X X VIM (carbapenemase) X X 24
23 25
24 BioFire FilmArray Blood Culture ID Panel Gram + Bacteria: Enterococcus spp. L. monocytogenes Staphylococcus spp. S. aureus Streptococcus spp. S. agalactiae (Group B) S. pyogenes (Group A) S. pneumoniae Antibiotic Resistance: meca Van A/B KPC Gram - Bacteria : A. baumannii Enterobacteriaceae Enterobacter cloacae Complex E. coli H. influenzae K. oxytoca K. pneumoniae N. meningitidis P. aeruginosa Proteus spp. S. marcescens Fungi: C. albicans C. glabrata C. krusei C. parapsiolosis C. tropicalis Identifies pathogens in 9 out of 10 positive blood cultures 26
25 Combining rapid pathogen ID with locally derived treatment guidelines, as application of these guidelines would have resulted in appropriate antimicrobial intervention for 99.2% of blood cultures growing organisms detected by the BCID panel. 27
26 Mortality Risk with Inappropriate Therapeutic Selection Abx P-T/Vanco BC+ Abx Dori AST K.pneumoniae Amikacin Ampicillin Cefazolin Cefoxitin Ceftazidime Ceftriaxone Cefepime Colistin Doripenem Ertapenem Gentamicin Imipenim Levofloxacin Meropenem Piperacillin/taxobactam Tigecycline Tobramycin R R R R R R R S R R R R R R R S R Time to critical AST result 13 hr: K. pneumoniae KPC+ 28
27 Power Ionising Vacuum Negative MALDI-TOF Mass Spectrometry 29
28 Off label use: Laboratory validated 30
29 Performance of Direct Testing 31
30 32
31 33
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33 35
34 Beta lactamase with cefotaxime Discrimination between E. coli strains that were R or S to aminopenicillins and R to 3rd-generation cephalosporins in Enterobacteriaceae that constitutively produced class C lactamases had a with a sensitivity and a specificity of 100%. Discrimination was more difficult in species expressing class A -lactamases, as these enzymes can generate false-positive results. Thus, the sensitivity and specificity for this group were 100% and 91.5%, respectively. Cefotaxime 36
35 Whole Genome Sequencing 37
36 Roche (Oxford) MinION Nanopore Sequencing 38
37 40
38 Rapid Direct Specimen Detection
39 Curetis Unyvero LRTI Panel 42
40 27 (55.1%) and 4 (8.2%) harbored multiple bacteria by UPA and culture, respectively. Single pathogen was detected in 8 patients (16.3%) by UPA and 4 patients (8.2%) by culture Thirteen different genes were detected from 38 patients, including ermb (40.8%), blaoxa-51-like (28.6%), sul1 (28.6%) int1 (20.4%) integrase, meca and blactx-m (12.3% each). Time from sample testing to results was 4 h (UPA) versus 48 to 96 h (culture) Initial empirical treatment was changed within 5 to 6 h in 33 (67.3%) patients based on UPA. Thirty (62.2%) of the patients improved clinically. Potential of a multiplex PCR-based assay for accurate and timely detection of etiological agents of NP, multidrug-resistant (MDR) organisms, and resistance markers, which can guide clinicians in making early antibiotic adjustments. 43
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42 T2 BioSystems Clinical Trial Data Blood specimens from 1801 hospitalized patients Overall specificity: 99.4% (95% CI, 99.1% 99.6%) with a mean time to negative result of 4.2 ± 0.9 hours 98.9% (95% CI, 98.3% 99.4%) for C. albicans/c. tropicalis 99.3% (95% CI, 98.7% 99.6%) for C. parapsilosis 99.9% (95% CI, 99.7% 100.0%) for C. krusei/c. glabrata Overall sensitivity: 91.1% (95% CI, 86.9% 94.2%) with a mean time of 4.4 ± 1.0 hours for detection and species identification 92.3% (95% CI, 85.4% 96.6%) for C. albicans/c. tropicalis 94.2% (95% CI, 84.1% 98.8%) for C. parapsilosis 88.1% (95% CI, 80.2% 93.7%) for C. krusei/c. glabrata LOD was 1 CFU/mL for C. tropicalis and C. krusei, 2 CFU/mL for C. albicans and C. glabrata, and 3 CFU/mL for C. parapsilosis NPV was estimated to range from 99.5% to 99.0% in a study population with 5% and 10% prevalence of candidemia, respectively 45
43 Rapid Susceptibility Advantage of speed: Rapid institution of appropriate therapy Antimicrobial stewardship programs Know the limitations: May be a preliminary result Genetic testing may miss new strains or variants (meca dropouts, mecc, carbapenemase genes) Phenotypic testing is still essential Understand the difficulties in standardization (MALDI) Teach: May tell you what you cannot treat with but may not tell you with what you can treat with 46
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