Etest Reading

Etest Reading

AB bioMrieux 2008

Etest Reading

Endpoint criteria relates to:

Technical issues Drug specific

Organism specific
Resistance mechanisms

Consult technical documents

Etest Reading Guides (ERG 001, 002) Customer Information Sheets (CIS 006, CIS 007) Etest Technical Guide (ETG 006) Mycobacteria

Molecule Size

Small molecule, larger ellipse

Large molecule, smaller ellipse

Basic Etest Reading

Read MIC where growth intersects the strip

If between markings, read upper MIC value

Between Markings

Read the upper value

First Reading Step Sharp Endpoints

Is it a sharp endpoint?

YES: then read MIC where growth intersects the strip. VA, MIC 1.0 g/mL

Second Reading Step Not a Sharp Endpoint

Is it a sharp endpoint?

NO: Is the drug bacteriostatic or bactericidal?

Rule: CIDAL DRUGS: Read at complete

inhibition of ALL GROWTH (haze, micro- and macrocolonies), Read 100% inhibition. MIC 0.064 g/mL

cidal

static

STATIC DRUGS: Read at the first

significant inhibition of growth. Ignore trailing and microcolonies. Read the socalled 80% inhibition. MIC 0.032 g/mL

Macrocolonies

Always read macrocolonies for both static and cidal drugs.

MIC 256 g/mL

Sharp or Hazy Endpoint?

MIC 1.5 g/mL

Sharp or Hazy Endpoint?

Bactericidal READ macrocolonies, microcolonies and trailing

MIC 32 g/mL

Sharp or Hazy Endpoint?

Bacteriostatic IGNORE trailing and microcolonies

MIC 0.032 g/mL

Sharp or Hazy Endpoint?

MIC 0.75 g/mL

Sharp or Hazy Endpoint?

Bactericidal

MIC 0.094 g/mL

Read microcolonies between 0.064-0.094

Sharp or Hazy Endpoint?

Bacteriostatic

MIC 0.25 g/mL

Read first significant inhibition of growth, 80% rule

Avoid too heavy an inoculum

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Ignore tunnel of growth along strip

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If uneven intersection - read upper value if 1 dilution

If > 1 dilution, test must be repeated

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Proteus - dont read the swarm!

18

Dont read haemolysis

19

Bactericidal Drugs Read at complete inhibition

Look for hazes, micro- and macrocolonies

-lactams Carbapenems Cephalosporins

Isoniazide Aminoglycosides

Quinolones
Metronidazole Rifampicin

Ethambutol
Glycopeptides Fosfomycin

Amphotericin B

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Pneumo - Pen G
Obtain good confluent growth Look for microcolonies and hazes MIC: 4.0 g/mL

21

Look for colonies in the dip

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Observe microcolonies from 8 to 32

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Paradoxical effect - beta-lactams

Induction of regrowth at higher concentrations of the antimicrobial Read the highest value

24

Bacteriostatic Drugs

Read at 80% inhibition i.e. first point of significant change as judged by the naked eye (CIS 007).

Tetracyclines Streptogramins Oxazolidinones

Lincomycins
Chloramphenicol Sulfonamides Trimethoprim

Macrolides
Azoles

Echinocandins

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80% for Bacteriostatic Drugs

Bacteriostatic drugs Endpoint rule: 80% inhibition MIC: 0.032 g/mL Comment: Read at the first point of significant inhibition as judged by the naked eye

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Linezolid

Static endpoint at 90% inhibition

MIC: 1 g/mL Trailing: 0.5 2 g/mL

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MRSA - low level heteroresistance

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Other Reading Rules

Pipercillin/tazobactam
Ticarcillin/clavulanic acid Ignore the inhibition of growth caused by the activity of the inhibitor (tazobactam, clavulanic acid) and extrapolate the ellipse to give the MIC of pipercillin or ticarcillin.

Echinocandins

Ignore regrowth at higher concentrations (paradoxical effect) for echinocandins

MIC: 0.125 g/mL

Distorted ellipse

Wet surface, repeat test

Interpretation of POSITIVE Etest ESBL

Cephalosporin with Clavulanic acid shows 3 log dilution lower than cephalosporin without Clavulanic acid; ratio of CT /CTL 8 Deformation of the ellipse Phantom zone All are Positive for ESBL See Etest ESBL Package Insert

ESBL POSITIVE

CT 0.5 and CT/CTL 8

or

Report:
ESBL producer and resistant to all penicillins, Cephalosporins and aztreonam (CLSI M100-S series).

TZ 1 and TZ/TZL 8
or

PM 0.25 and PM/PML 8

or

zone or deformation of the CT, TZ or PM ellipse

Phantom

ESBL

MIC: CT 1.5 & CTL 0.047 CT/CTL 1.5/0.047= 32 = ESBL+

ESBL +

Phantom zone = ESBL +

Deformation of ellipse = ESBL +

Categorisation of S, I, R

Use MIC breakpoints from most recent CLSI M100-S or own regulatory body (eg. EUCAST, BSAC) If not available, consult the litterature Etest gives values in-between 2-fold dilutions

Round-up to next upper dilution before S, I, R

Report the exact MIC and S, I, R

35

Interpreting 1/2 Dilution

S: 0.064 I: 0.125 - 1 R: 2 MIC 1.5 becomes 2 g/mL Category = R

R I S

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Technical Documents

Consult technical documents

Etest Reading Guides

ERG 001 antimicrobials
ERG 002 - antifungals

Customer Information Sheets

CIS 006 list of static/cidal drugs CIS 007 reading of sharp and hazy endpoints

Etest Technical Guide (ETG 006) Mycobacteria

Etest Reading Practice

AB bioMrieux 2008

FX code for: Cefoxitin Endpoint rule: 100% inhibition

MIC: 16 g/mL
Comment: Dont over-read

39

FX code for: Cefoxitin Endpoint rule: 100% inhibition MIC: 32 g/mL Comment: Macro-colonies = resistant subpopulation

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CM code for: Clindamycin

Endpoint rule: 80% inhibition MIC: 256 g/mL

Comment: Lawn of macrocolonies in discernable ellipse means resistant subpopulation

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MZ code for: Metronidazole Endpoint rule: 100% inhibition MIC: 0.75 g/mL Comment: Wide ellipse, sharp endpoint

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IP code for: Imipenem Endpoint rule: 100% inhibition

MIC: 0.064 g/mL

Comment: Wide ellipse, slight haze at endpoint

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AK code for: Amikacin Endpoint rule: 100% inhibition

MIC: 0.75 g/mL

Comment: Sharp endpoint

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CH code for: Clarithromycin Endpoint rule: 80% inhibition

MIC: 1 g/mL
Comment: Ignore hazes between 1-2

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TZ code for: Ceftazidime Endpoint rule: 100% inhibition MIC: 32 g/mL Comment: Read all microcolonies (SCVs) 6 - 32

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VA code for: Vancomycin

Endpoint rule: 100% inhibition

MIC: 1g/mL Comment: Read all growth

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EM code for: Erythromycin

Endpoint rule: 80% inhibition MIC: 0.064 g/mL

Comment: Ignore hazes bet. 0.064- 0.19

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CO code for: Colistin Endpoint rule: 100% inhibition

MIC: 0.19 g/mL

Comment: Ellipse tapers/ dips towards the endpoint

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IP code for: Imipenem Endpoint rule: 100% inhibition

MIC: 8 g/mL
Comment: Macrocolonies within ellipse, ignore line of growth along the strip

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OX code for: Oxacillin

Endpoint rule: 100% inhibition

MIC: 32 g/mL Comment: Read all growth (colonies in ellipse = mecA heteroresistance)
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LZ code for: Linezolid Endpoint rule: 90% inhibition MIC: 1 g/mL Comment: Ignore hazes between 1-2

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CD code for: Cefdinir (or other lactam) Endpoint rule: 100% inhibition MIC: 256 g/mL Comment: Paradoxical effect, regrowth 2- >256

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AT code for: Aztreonam Endpoint rule: 100% inhibition MIC: 3 g/mL Comment: Sharp endpoint

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MM code for: Mecillinam

Endpoint rule: 100% inhibition MIC: 256 g/mL

Comment: Macrocolonies in ellipse = resistant subpopulation

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CE code for: Cephalothin

Endpoint rule: 100% inhibition MIC: 96 g/mL

Comment: Small ellipse at high MIC

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TLc code for: Ticarcillin/clavulanic acid (2 g/ mL) Endpoint rule: 100% inhibition MIC: 2 g/mL Comment: Deformation of ellipse to straight dip 4 0.023 due to clav.acid activity Extrapolate ellipse curve to strip

57

TLc code for: Ticarcillin/clavulanic acid Endpoint rule: 100% inhibition MIC: 256 g/mL Comment: Resistance induced by clavulanic acid

58

DPC code for: Daptomycin (overlaid with constant level of calcium)

Endpoint rule: 100% inhibition

MIC: 1.5 g/mL

Comment: Sharp endpoint

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FL code for: Fluconazole Endpoint rule: 80% inhibition

MIC: 0.19 g/mL

Comment: Lawn of microcolonies in discernable ellipse, morphology change from 0.19

FL code for: Fluconazole Endpoint rule: 80% inhibition

MIC: 1 g/mL
Comment:

IT code for: Itraconazole Endpoint rule: 80% inhibition

MIC: 0.125 g/mL Comment: Clear endpoint

AP code for: Amphotericin B Endpoint rule: 100% inhibition MIC: 0.38 g/mL

Comment: Read all growth carefully! (microcolonies 0.1250.38)

FC code for: Flucytosine Endpoint rule: 90% inhibition MIC: 2 g/mL Comment: Ignore trailing, minute colonies above 2

FC code for: Flucytosine Endpoint rule: 90% inhibition MIC: 0.094 g/mL

Comment: Wide ellipse, ignore minute colonies above 0.094

CS code for: Caspofungin Endpoint rule: 80% inhibition MIC: 0.032 g/mL

Comment: Sharp endpoint

CS code for: Caspofungin Endpoint rule: 80% inhibition MIC: 0.125 g/mL

Comment: Sharp at 0.125, ignore regrowth of microcolonies 0.75 - 32