how can the laboratory detect carbapenemases has the best balance of sensitivity and specificity...
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You first
• Do you screen for carbapenemases Rectal screening
AST results only
• Do you use Chromogenic media for carbapenemase screens
• How many use the Modified Hodge Test?
• Do you perform any molecular confirmations on possible carbapenemases?
Outline
• Carbapenemases Brief description
Will not discuss epidemiology
• Detection methods Development of phenotypic methods
(Molecular methods)
(Novel techniques)
Carbapenems
• Broad spectrum of activity
• Stable to hydrolysis by most ‐lactamases
• Drugs of choice for treatment of infections caused by penicillin‐ or cephalosporin‐resistant GNB
• Resistance more common in nonfermenters Pseudomonas aeruginosa
Acinetobacter spp.
Carbapenem Resistance
Enterobacteriaceae
• Lack of drug penetration porin mutations
efflux pumps
• Acquisition of carbapenemase genes that encode enzymes capable of degrading carbapenems
Ambler class B metallo‐‐lactamases; NDM, VIM, IMP
Ambler class A serine enzymes; KPC
Ambler class D serine enzymes; OXA‐48 types
CarbapenemResistant
(R+I)
2012
Escherichia coli
Pseudomonas aeruginosa
http://www.ecdc.europa.eu/
Klebsiella pneumoniae
2010EARS-Net
Carbapenem screening criteria
Screening criteria Clinical breakpoints
Organism MIC (mg/L) ZD (mm) ECOFF (mg/L) a EUCAST CLSI
Meropenem E. coli, Klebsiella spp., Enterobacter spp., Salmonella spp. > 0.25 b < 25 0.125 ≤ 2 ≤ 1
Citrobacter spp., Serratia spp., Proteus spp., M. morganii, Providencia spp.
> 0.25 < 25 0.25 ≤ 2 ≤ 1
Pseudomonas spp., Acinetobacter spp. > 4 ≤ 26 2 ≤ 2 ≤ 2
The recommended screening criteria is meropenem MIC >0.125 mg/L; Vitek 2 and Phoenix card range is 0.25-16 mg/L. As such >0.25 mg/L is used.
Meropenem has the best balance of sensitivity and specificity
EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance. December 2013
CLSI M100 S24
Ambler class Enzyme
Hydrolysis profile
Inhibitor2GC 3GC AZT CAR
A
SME‐1 to ‐3 ‐ + + ++Clavulanate,tazobactam, sulbactam, NXL‐104
NmcA ‐ + ‐ ++
IMI‐2 ‐ + ‐ ++
GES‐4, ‐5, ‐6 + + ‐ +
KPC‐2 to ‐12 ‐ ++ + ++Clavulanate, tazobactam, boronicacid, sulbactam
BIMP, VIM ++ ++ ‐ ++ EDTA, thiol agents
(SMA/MPA)NDM ++ ++ ‐ +
D OXA‐48 like +/‐ +/‐ ‐ + NaCl
Nordman et al, (2012) Trends in Mol Med 18:263SMA = mercaptoacetic acidMPA = mercaptopropionic acid
Carbapenemase Detection Methods
• Imipenem hydrolysis (UV spectrophometric)• Use of various enzyme inhibitors
Combined Disc Tests Double Disc Synergy Tests
• Modified Hodge Test • MIC strips • Colorimetric Detection • Chromogenic Agars• Microdilution Tests • Detection of genes using molecular methods• Immunochromatography Assay• Imipenem hydrolysis (MALDI‐TOF mass spectrometry)
Algorithm for Phenotypic Detection of Carbapenemase
Synergy with BA AND cloxacillin
Synergy with DPA/EDTA only No synergy
KPC (or class A carbapenemase)
AmpC (chromosomal or plasmid-acquired)
plus porin loss
Metallo-β-lactamase (MBL)
ESBL + porin loss AND OXA-48
Meropenem10 µg ZD <=25 mmMIC > 0.12 mg/L
Synergy with BA
Interpretation of Phenotypic confirmation tests (carbapenemases in bold type)
β-lactamase
Synergya with meropenem(10 µg) disc/tablet and Temocillin
MIC > 32mg/L ZD <11 mm
DPA/ EDTA
APBA/ PBA
DPA+APBA CLOX
MBL (class B) Yes No No No Variable b
KPC (class A) No Yes No No variableMBL + KPC c v v Yes No variable
OXA-48-like (class D) No No No No YesAmpC + porin loss No (Yes) no (Yes) variable
ESBL + porin loss No No No No No
Abbreviations: DPA = dipicolinic acid; APBA = aminophenyl boronic acid; PBA = phenyl boronic acid; CLOX = cloxacillin
Tsakris A et al. JAC. 2010;65:1664
AmpC/ESBL
Three combined‐disc testβ‐lactamasedetected
PBA (400 µg)
EDTA(292 µg)
PBA+EDTA
Sensitivity Specificity
KPC + ‐ + 100% 98.5%
MBL ‐ + + 100% 100%
MBL/KPC ‐ ‐ + 96.8% 100%
KPC/VIM/ESBL KPC/ESBL VIM
Increase ≥ 5 mm
Carbapenemase Detection KitsMASTDISCS™ ID• Carbapenemase(Enterobacteriaceae) detection disc set (D70C) A Carbapenem 10 µg B Carbapenem 10 µg + MBL inhibitor
C Carbapenem 10 µg + KPC inhibitor
D Carbapenem 10 µg + AmpC inhibitor
[Temocillin 30 µg]
Rosco Diagnostica CE• KPC/MBL and OXA‐48 Confirm Kit (98015) MRP10 meropenem 10 µg MRPBO meropenem 10 µg + phenylboronic acid (AmpC/KPC inhibitor)
MRPCX meropenem 10 µg + (AmpC inhibitor)
MRPDP meropenem 10 µg + dipicolinic acid (MBL inhibitor)
Temocillin 30 µg
ROSCO Diagnostica CE
Total Metallo‐β‐lactamase Confirm Kit (98016)
McConkey Agar
The kit contains 5 tablets:• MRP10 Meropenem 10 µg
• DPA Dipicolinic acid
• IMI10 Imipenem 10 µg
• IM+DP Imipenem 10 µg + DPA
• IM10E Imipenem 10 µg + EDTA
MHA
MAC
ROSCO Diagnostica CE
KPC/MBL in P. aeruginosa/Acinetobacter Kit• uses two substrates to enhance the detection
The kit contains 5 tablets:• MRP10 meropenem (10 ug)• MRPBO meropenem (10 ug) +
phenylboronic acid (KPC and AmpCinhibitor)
• MRCXH meropenem (10 ug) + cloxacillin high (AmpC inhibitor)
• IMP10 imipenem (10 ug)• DPA dipicolinic acid
McConkey Agar
Disc Methods
• Availability of commercial discs/tests have helped to standardise methods
• Isolates with multiple resistance mechanisms may make interpretation difficult
• Optimum distance between discs for detection of synergy may vary for different countries/regions
• Changes to standard guidelines
• Stability of discs and inhibitors one manufacturer recommends 3 day shelf life for meropenem discs once cartridge opened!
“Convenient” DDST Test
• Imipenem based test with TE + MPA (1:320) most sensitive (100%) and specific (100%)
• TE alone did not detect 2 IMP‐1 A. baumanniiand 1 SPM‐1 P. aeruginosa
• MPA alone failed to detect 1 VIM‐2‐like P. aeruginosa
Kim et al. JCM (2007) 45: 2798
Convenient DDST Test – for MBLs
SMA 3 µl TE + MPA (1:320)
Tris-EDTAimipenem 10 µg10 cm edge to edge
Kim et al JCM 2007; 45: 2798
Modified Hodge Test
• CLSI‐recommended confirmatory test for carbapenemase production
• Lacks specificity
• Long turnaround time
• Poor specificity for MBL detection
Positive
negative
Positive
Enhanced growth of E. coli ATCC 25922
Detecting class B enzymes:MBL Etests
• imipenem (I) vs. imipenem + EDTA (IPI)• ratio 8 consistent with MBL production• zone distortion consistent with MBL production• sensitivity - good ; specificity - poor
MIC Strip Tests
• Etest® KPC MP/MPB Meropenem (0.25 μg/ml – 16 μg/ml) / Meropenem (0.064 μg/ml – 4 μg/ml) + boronic acid derivative (constant level)
• Liofilchem® MIC Test strip MRP/MBO Meropenem (0.25 μg/ml – 16 μg/ml) / Meropenem+Boronic acid (0.032 μg/ml –2 μg/ml)
• Liofilchem® MIC Test strip ETP/EBO Ertapenem (0.25 μg/ml – 16 μg/ml) / Ertapenem+Boronicacid (0.062 μg/ml –4 μg/ml)
• Several methodology changes since 1st
published Whole bacterial cells, rather then supernatant after lysis
Eppendorf tubes rather than micotitre tray
Increased concentration of imipenem
Dortet et al, JCM 2012; 50:3773-3776
Nordman et al, EID 2012; 18:1503-1507 Dortet et al, AAC 2012; 56:6437-6440
Dortet et al, JMM 2014; 63:772
Carba NP (Nordmann‐Poirel) Test
SOLUTIONS• Solution A: 0.05% phenol
red + 0.1 mM ZnSO4
• Solution A + imipenem (6 mg/mL)
Carba NP (Nordmann‐Poirel) TestREQUIREMENTS• 1.5 ml eppendorf tubes• 0.5% (w/v) phenol red solution• Imipenem‐cilastatin (MSD
Primaxin® 500)• 10 mM ZnSO4• 20 mM Tris‐HCl lysis buffer
B‐PER II or SoluLyse(ThermoFisher Scientific)
• Negative (wild‐type E. coli) and positive (K. pneumoniae OXA‐48 or K. pneumoniae KPC‐2) controls.
reconstitute imipenem in solution A each day of test
stable at RT for 1 week -20°C several months
Dortet et al, JMM 2014; 63:772
Carba NP (Nordmann‐Poirel) Test
PROTOCOL• Each isolate tested in paired tubes• Add 100 μl of 20 mM Tris‐HCl lysis buffer (B‐PERII) in each of
two 1.5 ml eppendorf tubes• Resuspend a 1/4 to 1/3 calibrated loop (10 μl) of bacterial
colonies in each tube
Can be taken directly from around carbapenem disc on MHA DO NOT use colonies from Drigalski or McConkey plates
• Vortex suspensions to ensure colonies well suspended• Add 100 μl of Solution A in the first tube and 100 μl Solution A
+ imipenem 6 mg/ml in the second tube, mix well• Incubate at 37°C for a maximum of 2 hours
1 ul loopful
Carba NP (Nordmann‐Poirel) Test
No antibiotic
Imipenem
Carbapenemase
Non producerRed Red
Carbapenemaseproducer
RedOrange/
Yellow
interpretable Yellow Yellow
Modifications/Adaptations• Blood cultures
2ml BHI + 70 mg/ml ZnSO4, no imipeneminoculated with 5 drops of positive blood culture into 2 ependorf tubes
Incubate at 37oC (agitated) for 3h
Centriguge at 10,000 g for 5 min
Perform Carba NP test on pellet
• CarbAcineto NP Test Acinetobacter spp. OXA‐types
Uses 5 M NaCl instead for PER bufferDortet et al, JCM 2014; 23 April
Blue‐Carba
• Carba NP variant
• Bromothymol blue as the indicator (pH 6.0 to 7.6)
Optimal pH range for most β‐lactamases
• No lysis buffer
• Direct colony approach
Pires et al, JCM 2013; 51:4281-4283
NDM
OXA-23
OXA-48
ATCC 25922
Blue‐Carba Test
• Test solution: 0.04% bromothymol blue (pH 6.0)
+ 0.1mmol/L ZnSO4
+ 3 mg/ml imipenem
Final pH 7.0
• Negative control solution• 0.04% bromothymol blue (pH 7.0)
• Colonies suspended directly into 100 ul test solution
Pires et al, JCM 2013; 51:4281-4283
NDM
OXA-23
OXA-48
ATCC 25922
Colorimetric Tests: Summary• 2 hour test, often within 15 minutes• High sensitivity (100%) and specificity often > 99%• CarbaNP: colour changes slower for VIM‐positive isolates and
isolates with low carbapenemMICs• Commercially available
Rapid CARB Screen Kit; Rosco Diagnostica 98021 Some reported difficulties in reading
• Modified procedure available for blood cultures• Modified procedure for Acinetobacter spp ‐ CarbAcineto NP
Uses 5M NaCl rather than PER buffer• Blue Carba
easier to perform and interpret; cheaper alternative to Carba NP Able to detect OXA‐48‐like enzymes
Vasoo et al, JCM 2013; 51:3097-3101 Hunag et al, JCM 2014; online 21 May 2014
ChromID ESBL
chromID Carba Brilliance CRE
chromID® CARBA SMART
OXA-48
Other CPE
CHROMagar KPC
Chromatic CRELiofilchem®
Chromogenic Media
MicroorganismCarbapenem‐resistant Klebsiella pneumoniaeCarbapenem‐resistant Escherichia coliCarbapenem‐resistant Enterobacter spp.Carbapenem‐resistant Citrobacter spp.Carbapenem‐resistant Non EnterobacteriaceaeOther microorganisms
Typical appearance of the coloniesBlue‐violetRedBlue‐greenBlue with red haloWhite to natural pigmentedInhibited
• Carbapenemase negative P. aeruginosa and Enterobacteriaceae isolates with reduced susceptibility to carbapenems (eg. AmpC with porinloss) can grow with direct screening,
• Enterococcus spp. and Candida spp. may also grow
Colours may difficult to discriminate
Published Evaluations
UK Protocols | P 8 | Issue no: 1.1 | Issue date: 08.05.14 | Page: 19 of 25 UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
General comments
• Predominance of a single type of carbapenemase
• different media may show different performances in different geographical locations
• It is likely that most methods have been optimised for KPC carbapenemases, as these predominate in several of the larger markets
• Enrichment broths supplemented with carbapenemshave also been advocated. inferior performance to commercially available chromogenic agars
extra day is required to obtain colonies for further testing
Molecular Methods
• Confirm the presence of carbapenemase genes
• Can be used for screening
• Mostly confined to research laboratories
• Only detect genes that are recognized by the repertoire of available probes Can miss detection of new enzymes
Mendes et al (2007) JCM 45;544
Multiplex Real‐Time PCR
blaIMP‐typeblaVIM‐typeblaGIM‐1blaSPM‐1blaSIM‐1
Genes detected
Metallo‐β‐lactamases
Molecular Methods
MCA using SYBR green
OXA-48-like and KPC multiplex
OXA-181OXA-48
KPC
IMP-4
IMP-14 VIM
IMP-7
IMP/VIM types multiplex
MicroArrays
• Detect over 100 specific DNA markers in one test
• Only ligated probes are amplified
• A small camera in the tube reader takes a picture of the array
• Analyzed using software
Check‐Direct CPE
• Real‐time PCR
• Directly from rectal or perianal swabs
• Results in 2 hours
• KPC, OXA‐48, or VIM/NDM
• Check‐MDR CT103XL OXA‐23, ‐OXA‐24, OXA‐58, GES, SPM, GIM and ESBLs (GES, PER, VEB, BEL)
Immunochromatography (IC) Assay
• Quick Chaser® IMP
• Detects all known IMP‐subtypes (IMP‐1 to ‐44)
• Enterobacteriaceae and NF‐GNB
Notake et al, JCM 2013; 51:1762-1768Kitao et al, JMM 2011; 87:330-337
Interpretation ~ 20 min
¥1,000 per test¥1,000 per test
Other Approaches
• Whole genomic sequence data
• MALDI‐TOF Mass Spectrometry
• Line probe assay AID, Germany; (ESBL and KPC)
Stoesser et al JAC May 2013
Van Belkum et al JCM (2013) 51:2018-2022
Bloemberg et al JAC Sep 2013
Summary• Need for prompt detection to limit spread
Essential for implementation of infection control procedures
May provide guidance for individual patient management
• Numerous detection methods now available. Chromogenicagars have improved and Mass Spectrometry is now available in many institutions
• Many organism have multiple enzymes and resistance mechanisms
• Be aware of changes to standard guidelines
• Although not mandatory, all laboratories are advised by the DoH to screen for carbapenemases
• The Blue Carba or CarbaNP can easily be incorporated into the routine workflow
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