conventional vs newly developed techniques for escmid

Conventional vs newly developed techniques for identification of resistance

Laurent POIREL

Medical and Molecular Microbiology Unit Dept of Medicine

University of Fribourg Switzerland

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ANTIMICROBIAL AGENT

MICRO-ORGANISM PATIENT ESCMID Online Lectu

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Antibiogram Reading

Ø Inh. zone (mm)

MIC (mg/L)

S-I-R ESCMID Online Lecture Library

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Clinical Categories

• CLSI (NCCLS): Clinical and Laboratory Standards Institute • EUCAST: European Committee on Antimicrobial Susceptibility Testing

• SFM: Société Française de Microbiologie

• BSAC: British Society for Antimicrobial Chemotherapy • MENSURA: Mesa Española de Normalización de la Sensibilidad y Resistencia a los Antimicrobianos

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“Interpretative reading of the antibiogram”

1.- Define phenotype of susceptibility and resistance 2.- Deduce the possible mechanism of resistance 3.- Adequate clinical categories to the inferred mechanism of resistance, and change phenotype if necessary


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S-I-R Microbiological knowledge

CLINICAL REPORT OF RESULTS

“Interpretative reading of the antibiogram”


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AES

S-I-R SOFTWARE

(Expert system)

CLINICAL REPORT OF RESULTS


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INTERPRETATION


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• Identification of the micro-organism (at the species level)

• Analysis of susceptibility/resistance phenotype - Groups (families) of antimicrobial agents - Antimicrobial indicators of resistance

• Define Phenotype - Common phenotypes - Unusual phenotypes - “Impossible” phenotypes

• Deduce biochemical mechanism of resistance • Clinical relevance of the inferred resistance • Re-define clinical categories

Interpreative reading


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E. coli

Wild-type phenotype (ß-lactams)

AMX

TIC

PIP

TZP

CF

CTT

CXM

FOX CTX

FEP MOX

CAZ AMC

IPM ATM

TCC


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E. coli Acquired penicillinase

AMX

TIC

PIP

TZP

CF

CTT

CXM

FOX CTX

FEP MOX

CAZ AMC

IPM ATM

TCC

?

K. pneumoniae

or


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E. coli AMX

TIC

PIP

TZP

CF

CTT

CXM

FOX CTX

FEP MOX

CAZ AMC

IPM ATM

TCC

High-level penicillinase


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Requirements of interpretative reading of the antibiogram

• Identification of the microorganism

• Analysis of S/I/R phenotype

• Use of indicator agents

• Study antibiotic-inhibitor combinations

• Quantitative study of susceptibility

• Use of high inocula (in some cases)

• Local epidemiology information

• Availability of reference methods


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Microorganism Identification

+ antibiogram

REPORT

Antibiogram Interpretation

Deduce Phenotype of Resistance

Deduce Biochemical Mechanism of Resistance

Clinical Relevance of the mechanism of

resistance

Re-Define Clinical Categories, if necessary Deduce susceptibility/resistance to non tested antimicrobial agents


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Livermore et al., JAC 2001


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Limitations to interpretative reading of the antibiogram

• High complexity of resistance mechanisms

• Limited information about some mechanisms of resistance

• Low level resistance

• Multifactorial multiresistance

• Oversimplification of “interpretative reading”

• Mistakes when deducing mechanisms of resistance


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Benefits of the interpretative reading of the antibiogram

• Adequacy of antimicrobial therapy

• Detection of new mechanisms of resistence

• Analysis of epidemiology of resistance

• Antimicrobial policy

• Improved quality in laboratory testing

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How to detect carbapenemases ?


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Question ; any carbapenemase here ?

E. coli

K. pneumoniae K. pneumoniae K. pneumoniae

E. coli E. coli

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM


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Nordmann et al. Clin Microbiol Infect 2012; 18:432-38

The reasons of the complexity


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Susceptibility testing : imipenem, ertapenem, meropenem: CLSI, EUCAST guidelines

Phenotypic detection - Hodge test; modified Hodge test - Inhibition; EDTA, clavulanic acid, boronic acid… Carbapenem hydrolysis (UV spectrophotometry, Mass spectro) Molecular biology - Specific PCR , multiplex PCR +/- sequencing - Real time PCR - DNA Microarray

Detection of carbapenemase producers in infected samples


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Infections

D 2

D 0

Blood cultures Urine Other samples

D 1 +

+

Antibiogram

Antibiogram


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Clinical breakpoints and screening cut-off values for carbapenemase-producing Enterobacteriaceae

When to suspect production of a carbapenemase ?


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Principal - KPC inhibited by boronic acid or clavulanic acid - MBL inhibited by EDTA or dipicolinic acid

Tests available - Combined Test (ROSCO) : meropenem +/- cloxacillin or dipicolinic acid or boronic acid - E-test MBL - Inhibition by EDTA (« home-made » technique)

Imipenem + EDTA Imipenem alone

Inhibition tests


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Negative control

Positive control

Modified Hodge test

Strain to be tested


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AmpC

C(–)

C(+)

AmpC

C(+)

CTX-M-15

CTX-M-15

CTX-M-15

- Low sensitivity with no specificity for different carbapenemases - Variable results with different carbapenems - False negative results: - weak results with certain MBLs (NDM) (increased sensitivity with ZnSO4 ) - False positive results: - CTX-M-15 or AmpC hyperproduction

Modified Hodge test


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NDM-1 (c)

OXA-48

T-

NDM-1 (a)

NDM-1 (b)

KPC-2

MH

OXA-48

T-

NDM-1 (a)

NDM-1 (b)

NDM-1 ( c )

KPC-2

MH + ZnSO4 (100 µg/ml)

Value of the Modified Hodge test for detection of emerging carbapenemases in Enterobacteriaceae

Girlich D, Poirel L, Nordmann P, J Clin Microbiol. 2011


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CAZ EDTA IMP

Disk diffusion synergy test: IMP + EDTA or CAZ + EDTA


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Combined-disk tests

IMP + EDTA

IMP

AMC

ATM ESCMID Online Lecture Library

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Disk combination test: carbapenem + boronic ac.

IMP IMP + boronicac. boronic ac.

Disk diffusion synergy test: IMP + boronic ac.

KPC: synergy with boronic acid


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Another basic and useful method (for expert labs)

• Measurement of carbapenem hydrolysis by UV spectrophotometry

- 10 µl of bacterial crude extract + 100 µM of imipenem - wavelength: 297 nm

Bernabeu, Poirel & Nordmann, DMID 2012


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Protocol :

1) Broth culture with the strain to be tested + carbapenem : 3-6h 2) Mass spectrometry

Mass spectrometry : MALDI-TOF

Carbapenem Carbapenem hydrolysis product

3) if carbapenemase + : hydrolysis of the carbapenem molecule leading to a degradation product

Advantages :

NDM-1

IMP-1

Specific / sensitive Fastness + Cheap if you dispose from the machine ! Disadvantages Material price Expertise

Hrabák et al. JCM. 2011 Burckhardt et al. JCM. 2011 Hrabák et al. JCM. 2012


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• Real-Time PCR :

- Check-MDR Real-Time PCR - Detect the presence of the carbapenemase gene - 4-5 h - Cost +++

• Specific PCR +/- sequencing :

- OXA-48-like / KPC / VIM / IMP / NDM - 3 to 5 h - Expertise ++ - Cost +

Molecular biology : PCR-based Techniques


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3 multiplex reactions: #1: blaIMP, blaVIM, blaSPM

#2: blaNDM, blaKPC, blaBIC, blaOXA-48

#3: blaAIM, blaGIM, blaSIM, blaDIM Poirel et al. Diagn Microbiol Infect Dis 2011; 70:119-23 - real time PCR for non-MBL: blaGES, blaIMI/NMC, blaKPC, blaOXA-48, blaSME Swayne et al. Int J Antimicrob Chemother 2011; 38:35-8

Molecular approaches

- simple and multiplex PCR + sequencing


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PCR + MASS SPECTROMETRY

PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS) Main application for identification of micro-organisms

Possible for resistance mechanisms : -mecA, mupA, ermA, ermC -vanA et vanB -blaKPC -INH et RIF (MTB) -FQ (gyrA, parC)

Desalting

Ecker et al., Nat Rev Microbiol 2008;6:553-6 Lavigne et al., Clin Chem Lab Med 2012;0:1-14

Multiplexing

Analysis by mass-spec ESCMID Online Lectu

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www.check-points.com Naas et al. J Clin Microbiol 2011; 49:1608-13 Willemsen et al. J Clin Microbiol 2011; 49:2985-7 Bogaerts et al. Antimicrob Agents Chemother 2011; 55:4457-60

Molecular tests: commercial microarrays

- colony: sensitivity and specificity of 100% for KPC, OXA-48 and MBLs - clinical samples: not yet evaluated


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Next step automation, multiplexation


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Vortex and Dispense Sample into Port S

2

Insert Cartridge and Start Assay

3

Insert Swab into Elution Reagent Vial and Break at Score

1 Total Hands-On time <1 Minute

No Specialized Training Required to Achieve Reliable, Reproducible Results

INTEGRATED PLATFORM AND TEST


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Xpert MDRO Cartridge

Cartridge detects three carbapenem resistance gene families (54 genes in total) –blaKPC –blaNDM –blaVIM • Sample : Rectal Swabs • Result in 47 minutes


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MRSA Surveillance SA Nasal Complete C. diff & C. diff/Epi vanA for VRE MRSA/SA SSTI MRSA/SA Blood Culture CT/NG EV Flu GBS & GBS Lim Broth Factor II&V MTB/RIF

2013

2014 2015-16 14 Tests

18 Tests

27 Tests

16 Tests

25 Tests

36 Tests

US

Intl

Updated Targeted Xpert® Test Menu

MRSA Surveillance SA Nasal Complete C. diff/epi vanA/vanB VRE MRSA/SA SSTI MRSA Blood Culture EV Flu GBS Factor II&V BCR/ABL v1 & BCR/ABL v2* CT & CT/NG HPV* MTB/RIF

Norovirus CARBA-R (MDRO) Trichomonas CT/NG LBC HIV Viral Load HIV Qualitative HCV Viral Load Flu/RSV Bladder Monitor/Sympto

HSV 1/2 Typing CT/NG LBC Vaginitis CARBA-R (MDRO) HIV Viral Load HCV Viral Load Bladder Monitor Bladder Symptomatic CW Flu/RSV

Trichomonas Norovirus Flu/RSV BCR/ABL v2

42 Tests

37 Tests

2017-18 HPV HBV Viral Load Group A Strep Sepsis Fungal Meningitis/Encephalitis Respiratory Panel Gastro Panel OncoScreen Lung Prostate Recurrence Risk Breast Recurrence Sig Breast Therapy Stratifier

CW CT/NG CW Vag CW GBS CW GAS

Targeted Test Menu Subject to Revisio

HBV Viral Load HSV 1/2 Typing Vaginitis Group A Strep Sepsis Fungal Respiratory Panel Gastro Panel OncoScreen Lung Prostate Recurrence Risk Breast Recurrence Sig Breast Therapy Stratifier

Meningitis/Encephalitis


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The Carba NP test

N O

R

COOH

S-R

Carbapenems

Imipenem Meropenem Ertapenem Doripenem

H2N

R

COOH

S-R O

HO

Acid production

Carbapenemase

pH

Colorimetric detection ESCMID Online Lecture Library

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The Carba NP test; the kit

96 wells plate

Diluted red phenol pH=7.8 + ZnSO4 0.1 mM

Lysis buffer

3 mg of imipenem powder ESCMID Online Lecture Library

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Results

E. coli VIM-1

K. pneumoniae CTX-M-15 + impermeability

K. pneumoniae OXA-48

K. pneumoniae KPC-2

E. coli NDM-1

E. coli IMP-1

Carbapenem - +

Yellow = carbapenem hydrolysis


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Ambler class Carbapenemase type Mean time for

positivity

A KPC 15 min- 1h

A GES-2, -5 1h-1h30

B NDM 20-50 min

B VIM 20-50 min

B IMP 5-30 min

D OXA-48 30-40 min

The Carba NP test


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Question ; any carbapenemase here ?

E. coli

K. pneumoniae K. pneumoniae K. pneumoniae

E. coli E. coli

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM


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Question : any carbapenemase here ?

E. coli VIM-1

K. pneumoniae CTX-M15 + impermeability K. pneumoniae OXA-48 K. pneumoniae KPC-2

E. coli NDM-1 E. coli IMP-1

(+) 30 min (-) 2 hours (+) 5 min

(+)25 min (+) 30 min (+) 20 min


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The Carba NP test

1- Rapid; less than 2 h

2- Sensitive; 98% (1,600 tested strains, French

National Reference Center)

3- Specific: 100%

4- Detection of any type carbapenemase activity

5- Cheap : 0.5 euro

6- Easy-to-handle

7- Implementable worldwide


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Principal - ESBLs inhibited by clavulanic acid or tazobactam - Double-disk synergy test - E-test CAZ / CAZ + clav., or FEP / FEP + clav.

Inhibition tests


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Detection of ESBLs : ESBL NDP test

N O

R

Cefotaxime

H2N

R

COOH

R’

O

HO

Production of acid

ESBL

pH

S

COOH

R’

S’

+ tazobactam

Nordmann, Dortet, Poirel. 2012. J. Clin. Microbiol.

N O

R S

COOH

R’ ESCMID Online Lectu

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Infections

D 2

D 0

Blood cultures Urine Other samples

D 1 +

+

Antibiogram

Antibiogram

Carriage = stools

ESBL NDP test


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High-throughput sequencing

Dune et al., Eur J Clin Microbiol Infect Dis 2012;31:1719-26

Theoritically, will allow : - accurate identification - obtention of a « virtual » antibiogram - typing (criteria to be defined)

Preparation of the sample is critical Gene expression level Cost


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TRANSCRIPTOME BY RNA-SEQ

Febrer et al., Trends Biotechnol

Sample preparation is critical and difficult Quite long process bio-informatical analysis Costly

Robust and reproducible method HOWEVER…


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Conclusion

- Increased prevalence of carbapenemase

producers worldwide.

- Very few novel antibiotics will be launched within the next years.

- Diagnostic techniques are now available for accurate diagnostic of carbapenemase producers

- Two main goals: - - Carbapenem stewarship - - Outbreak prevention and control


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CONCLUSION

Methods

Phenotypic Inhibition of bacterial growth

Current reference May be fastened

(micro-fluidic, nanotechnologies)

Fastness/Reliability ?

Genotypic Direct detection of resistance

Multiplexable Bio-informatic

Hetero-resistance?

Unknown mechanisms ?

Complementary approaches mandatory


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conventional vs newly developed techniques for escmid

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