Antifungal Susceptibility Testing

37 Pages·2017·1.09 MB·English

y
r
a
r
b
i
L
r
e
Antifungal Susceptibility Testing (AFST)
o

h
D
EUCAST or commercial methods - which method to use?
t
I u
M
Maiken Cavling Arendrup,
a
Prof, DMSci, MD, PhD
C
EUCyAST development laboratory for fungi
S Statens Serum Institut
b
Rigshospitalet
E University of Copenhagen
©
Copenhagen, Denmark

[email protected]

Disclosures:

Research grants/contract work (Paid to SSI): Amplyx, Basilea, Cidara, F2G, Gilead, Pfizer & T2Candida
Speaker honoraria: Astellas, Basilea, Gilead, MSD, Novartis, Pfizer & T2Candida
Chair(wo)man for EUCAST-AFST
Past advisory board (≤ 2014): MSD, Pcovery, Pfizer
M Cavling ARENDRUP
y
r
a
Factors to consider when implementing AFST in your lab
r
b
Methods i
L
Reference microbroth dilution
r
e
SensiTitre microbroth dilution
o
Gradient strip agar diffu sion e.g. Etest Liophilchem
h
D
Semi-automated methods e.g. Vitek
t
I u
Different compoundMs
Different reference
a
…. different behaviour
breakpoints & QC ranges
C
Echinocandins
EUCAST
y
Azoles
CLSI
S
b
Amphotericin B
E 5-FC
© Different expertise
Centre characteristics ?
Separate mycology trained staff?

Yeast only/and moulds? Which AST tests for bacterial testing

No. of isolates are already implemented?

Underlying patient population

Local epidemiology

M Cavling ARENDRUP
y
r
a
Factors to consider when implementing AFST in your lab
r
b
Methods i
L
Reference microbroth dilution
r
e
SensiTitre microbroth dilution
o
Gradient strip agar diffu sion e.g. Etest Liophilchem
h
D
Semi-automated methods e.g. Vitek
t
I u
Different compoundMs
Different reference
a
…. different behaviour
breakpoints & QC ranges
C
Echinocandins
EUCAST
y
Azoles
CLSI
S
b
Amphotericin B
E 5-FC
© Different expertise
Centre characteristics ?
Separate mycology trained staff?

Yeast only/and moulds? Which AST tests for bacterial testing

No. of isolates are already implemented?

Underlying patient population

Local epidemiology

M Cavling ARENDRUP
y
r
a
Agenda
r
b
i
EUCAST Reference Test method review L
r
 Yeast AFST: E.Def 7.3 e
o
 Mould AFST: E.Def 9.3

h
 Azole screening agar for azole reD sistant A. fumigatus
t
I u
Commercial tests M
a
 Potential caveats associated with performance studies
C
 Issues concerning adoption of reference method BPs (EUCAST /CLSI) 
y
implication for SIR categorisation
S
b
‒ Echinocandins Vitek Semi-automated method
‒E Amphotericin B Etest gradient strips
examples
©
‒ Fluconazole SensiTitre microbroth dilution

My schematic comparison of AFST methods available

Check list when (before) implementing AFST

M Cavling ARENDRUP
y
r
a
Test method review: yeast EUCAST E.DEF 7.3.1 (2017)
r
b
i
Flat bottom plates IncuLbation at 35 ± 2˚C
r
RPMI with 2% glucose & MOPS e24h and  OD ↑ by ≥ 0.2
o
0.5-2.5 x 105 cells/mL 50% inhibition, Spec. reading
h
D
t
I u
Drug dilutions M P N
0.8
a
0.7 PSC
C
0.6 ANI
y
0.5 VOR
S e
b u
la
v 0.4 ITR
D
E O
0.3
FLU
©
0.2
Pos contr
0.1
50%
inhibition
0.0

Concentration mg/L

M Cavling ARENDRUP
y
r
a
EUCAST breakpoints (BP) Yeast
r
b
i
L
r
C. albicans C. glabratae C. krusei C. parapsilosis C. tropicalis
o
S ≤ R > S ≤ R > S ≤ R > S ≤ R > S ≤ R >

Amphotericin B EUCAST 1 1 1 1 1 1 1 h 1 1 1
D
CLSI 1 1 1 1 1 1 1 1 1 1
t
Anidulafungin EUCAST 0.03 0.03 0.06 0.06 0.06 0.06 0.002 4 0.06 0.06
I u
CLSI 0.25M 0.5 0.12 0.25 0.25 0.5 2 4 0.25 0.5
a
Caspofungin EUCAST nd nd nd nd nd nd nd nd nd nd
CLSI 0.25 0.5 0.12 0.25 0.25 0.5 2 4 0.25 0.5
C
Fluconazole EUCAST 2 4 0.002 32y - - 2 4 2 4
CLSI 2 4 0.002 32 - - 2 4 2 4
S
b
Itraconazole EUCAST 0.06 0.06 IE IE IE IE 0.12 0.12 0.12 0.12
E CLSI 0.12 0.5 0.12 0.5 0.12 0.5 0.12 0.5 0.12 0.5
©
Micafungin EUCAST 0.016 0.016 0.03 0.03 IE IE 0.002 2 IE IE

CLSI 0.25 0.5 0.06 0.125 0.25 0.5 2 4 0.25 0.5

Posaconazole EUCAST 0.06 0.06 IE IE IE IE 0.06 0.06 0.06 0.06

CLSI nd nd nd nd nd nd nd nd nd nd

Voriconazole EUCAST 0.12 0.12 IE IE IE IE 0.12 0.12 0.12 0.12

CLSI 0.12 0.5 IE IE 0.5 1 0.12 0.5 0.12 0.5

www.eucast.org M Cavling ARENDRUP
y
r
a
EUCAST Fluconazole MIC & outcome
r
b
i
L
Fluconazole, 258 cases
r
e
(128 candidaemia 58% C. albicans; 133 OPC all C. albicans) o

h
D
Candidaemia OPC ≥ 100 mg/d All
MIC in
t
mg/L I u
No. cure/Total % response No. cure/Total % response % response
M
a
< 0.5 98/107 92 26/26 100 93
C
y
S
1 6/6 100 4/4 100 100
S
b
2 1/1 100 1/1 100 100
E
©
4 3/3 100 5/9 56 67 I/SDD

8 2/5 40 7/32 22 24

R

≥16 3/4 75 0/60 0 5

Rodriguez-Tudela AAC 2007
M Cavling ARENDRUP
y
r
a
EUCAST echinocandin testing: separation WT >< R
r
b
i
L
r
e
o
C. albicans C. glabrata C. krusei C. parapsilosis C. tropicalis

10 C. albicans (S) 10 C. glabrata (S) 1D2 C. krusei (S) 8 C. parahpsilosis (S) 10 C. tropicalis (S)
9 9 9
C. albicans (R) C. glabrata (R) 10 C. krusei (R) 7 C. tropicalis (R)
8 8 8
6
A No. of isolates 34567 No. of isolates 34567 M I No. of isolates 468 No. of isolates 345 u t No. of isolates 34567
N a2
2 2 2
2
1 1 1 1
I 0 0 0 0 0
0.01 0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 C 0.01 0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 0.01 0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 0.01 0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 0.01 0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16
y
MIC (μg/ml) MIC (μg/ml) MIC (μg/ml) MIC (μg/ml) MIC (μg/ml)
S
b
E
M
10 C. albicans (S) 10 C. g©labrata (S) 12 C. krusei (S) 10 12 C. tropicalis (S)
I 9 C. albicans (R) 9 C. glabrata (R) 1101 C. krusei (R) 9 C. parapsilosis (S) 1101 C. tropicalis (R)
8 8 8
9 9
AC No. of isolates 34567 No. of isolates 34567 No. of isolates 45678 No. of isolates 34567 No. of isolates 45678
3 3
2 2 2
2 2
1 1 1 1 1
0 0 0 0 0
0.01 0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 0.01 0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 0.01 0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 0.01 0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 0.01 0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16
MIC (μg/ml) MIC (μg/ml) MIC (μg/ml) MIC (μg/ml) MIC (μg/ml)

Arendrup et al AAC 2010, Arendrup CMI 2012, Arendrup DRU 2013 & Mycoses 2014
M Cavling ARENDRUP
y
r
a
Test method review: Mould EUCAST E.Def 9.3.1 (2017)
r
b
i
Flat bottom plates L
r
e
RPMI with 2% glucose & MOPS
Amb Casp Vor oItra Flu Cnt

8-0.06 4-0.03 16-0.125
Inoculum 1-2.5 x 105 cells/mL h
D
(avoid clumps, standardisation by conidia
t
I D u
counting/spectrophotometre) r
M u
g
a
Incubation: 48 h  adequate growth* d
i
l
u
C
(*24 h may be sufficient for some t
i
y o
n
mucorales, some moulds require 72 h)
S s
b
Visual endpoint reading
E
©
Amphotericin & azoles =

no growth endpoint (M IC)

Echinocandins =

aberrant gro wth (MEC)
MIC/MEC 0.5 ≤0.06 0.5 0.5 >16

www.eucast.org M Cavling ARENDRUP
y
r
a
Visual reading – Moulds - Azoles & amphotericin B
r
b
Growth control column
D i
r NotLes
u
g
r
d e - Place a black paper on a white table
i
l o
u
it - Hold the plate elevated above the
o
h
n D
s table
t
I - Examineu sharpness of the line
M
through the wells
a
- Last well with a sharp line ~ the MIC
C
y
S
b
E
©

Mfg Psc Vrc Ivc Itc +ve

M Cavling ARENDRUP