Methods of Detecting reduced susceptibility to glycopeptides in S.

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BSAC Antimicrobial Susceptibility Testing & resistance
Meeting, 13th May 2010
Methods of Detecting reduced
susceptibility to glycopeptides in S.
aureus
Methods of Detecting Reduced Susceptibility to
Presenter:
Mandy
Wootton
Glycopeptides
in S. aureus
Phenotype
• Glycopeptide Resistant Staphylococcus
aureus GRSA
– vanA gene present, MIC VAN >32mg/L
• Glycopeptide Intermediate Staphylococcus
aureus GISA
– homogeneous resistance, mechanism unknown - MIC VAN
~4/8mg/L (Mu50)
• heterogeneous-Glycopeptide Intermediate
Staphylococcus aureus hGISA
– heterogeneous resistance, mechanism unknown - MIC VAN
~1.5/4mg/L (Mu3). Precursor to GISA
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
Prevalence
GRSA
• < 30 world wide
• None in UK
GISA/hGISA
• UK prevalence:
– <1% (EARSS)
• 6064 isolates (1 Intermediate)
• MIC
– 0% (2001-6 BSAC Bacteraemia)
• 615 isolates
• MIC
• Cardiff:
– 0.8%
• 669 MRSA
• PHX, disc, MHA5T, PAP-AUC
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
BSAC Breakpoints
MIC
<0.25
0.5
GSSA
1
2
hGISA
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
4
8
GISA
16
EUCAST Breakpoints
MIC
<0.25
0.5
GSSA
1
2
4
hGISA
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
8
GISA
16
Disc Diffusion 1
• ISA with 1:10 of 0.5 McFarland
inoculum
• 5g Vancomycin disc
• Incubation at 35-37°C for 1820h
• Interpretive Criteria:
Resistance
Susceptible
- 14mm
- 15mm
GRSA: Detected
GISA: NOT Reliably Detected
hGISA: NOT Detected
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
Disc Diffusion 2
Separation of phenotypes
30
Zone Diammeter (mm)
• ISA with 1:10 of 0.5
McFarland inoculum
• 5g, 30g Vancomycin disc
• 5g, 10g, 20g, 30g
Teicoplanin
• Incubation at 35-37°C for 1820h
GSSA
hGISA
GISA
20
10
0
V5
V30
• Low concn discs of V and T
differentiate GISA & GSSA
• None accurate for ID of hGISA
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
T5
T10
T20
Disc Concentration
T30
MIC Determination 1
• Standard Etest
–0.5 McFarland on ISA, 18-20h
–BPs 2 (S)/ >2 (I/R)
3
6
2
0
0
0
0
%
Correctly
Classified
100%
0
0
1
41
7
1
0
16%
0
0
0
2
8
0
7
88%
0.5 0.75
MRSA/GSSA
x10
hGISA
x50
GISA
x17
MIC
1
2
4
6
8
<0.25
0.5
GSSA
GRSA Detected
GISA Mostly
Detected
hGISA mostly
missed
1
2
4
hGISA
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
8
GISA
16
MIC Determination 2
Automated (PHX)
MRSA/GSSA
x22 repeats
• Repeat 1 MRSA/GSSA x22
GISA
• GSSA x10
hGISA x10
GISA x10
MRSA/GSSA
x10
hGISA
x10
GISA
x10
1
2
4
8
9
1
0
0
% Correctly
Classified
100%
0
6
4
0
40%
0
5
4
1
50%
1
2
4
18
4
0
hGISA
PAPAUC
PHX
V
MIC
Etest
V
MIC
PAPAUC
3.01
1.33
1.32
1.47
1.4
1.31
1.32
1.32
1.57
1.41
2
2
2
2
8
4
2
4
4
4
4
4
4
4
8
4
4
4
4
8
1
0.94
1.14
1.23
1.04
0.94
0.97
1.29
1.1
1.14
GSSA
PHX Etest
V
V
MIC MIC
4
2
4
4
2
2
2
2
4
2
%
Correctly
Classified
100%
4
2
2
2
2
2
2
2
2
2
PAPAUC
0.58
0.74
0.68
0.73
0.73
0.74
0.45
0.58
0.65
0.62
PHX Etest
V
V
MIC MIC
≤1
≤1
≤1
≤1
≤1
≤1
≤1
2
≤1
≤1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Some GISA & hGISA
identified
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
Summary
• Disc
– Detects GRSA (vanA)
– Some detection of GISA
– No reliable detection of hGISA
• MIC (Etest & automated)
– Detects GRSA
– Detects some GISA
– Detects few hGISA
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
Macro Etest
• 2 McF inoculum
• Brain Heart Infusion
• 48h incubation at 35-37°C in air
• Interpretative Criteria: (NOT MICs)
GISA or hGISA if 8mg/L plus 8mg/L
Vancomycin + Teicoplanin or 12mg
Teicoplanin alone.
Detects most hGISA and GISA
No distinction between GISA and hGISA
2% false positive
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
GRD Etest
•
•
•
0.5 McFarland inoculum
Mueller Hinton + blood
48h incubation at 3537°C in air
–
•
Reading at 24h and 48h
Interpretive criteria:
GISA or hGISA if VA
or TP 8mg/L
1)
2)
GISA if GRD+ and standard VA
MIC 4mg/L
hGISA if GRD+ and standard
VA MIC <4mg/L
MRSA/GSSA
x50 repeats
hGISA
x51
GISA
X26
% Correctly
Classified
No +
at
24h
0
No +
at
48
1
30
41
80.4%
24
26
100%
98%
Detects all GISA, majority at
24h
Detects most hGISA and GISA
2% false positives
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
Screening Agar 1
•
Mueller Hinton Agar + 5mg/L TP
(MHA5T)
• Multi-centre study
– 12 labs (Europe & US)
– 48 strains
– 3 method
– 10ul of 0.5 McF inoculum
– 48h Incubation at 35-37°C in
air
– Interpretive criteria:
hGISA or GISA if ≥1 cols
after 48hr
•
Brain Heart Infusion Agar +
6mg/L VA (BHI6V)
– 10μl of 0.5McF inoculum
– 24h Incubation at 35-37°C in
air
– Interpretive criteria:
hGISA or GISA if ≥2 cols
after 24hr
BHIA6V
MHA5T
Macro Etest
% GSSA
Correctly
Classified
97.4%
76%
98%
% hGISA
Correctly
Classified
11.5%
78.7%
70.3%
% GISA
Correctly
Classified
58.9%
93.8%
97.4%
* MHA5T: false positives reduced when 2 cols at
48h used
MHA5T Detects Most GISA
MHA5T Detects Most hGISA
Up to 24% false positives
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
Population Analysis Profile
– Area Under Curve 1
• “Gold Standard”
• 2 plates of BHIA + 0, 0.5, 1, 2, 2.5 & 4mg/L VA
• Overnight cultures of test organism and Mu3 in
TSB - diluted 1:10-3, 1:10-6
• Incubation at 35-37°C in air for 48hrs
106 / 103
106 / 103
106 / 103
103 / N
103 / N
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
103 / N
• Log viable count
vs. VA concn
graph
• Ratio = AUC of
test isolate/AUC
of Mu3
10 1 0
Viable count Log cfu/ml
PAP-AUC 2
PAP of Mu 3 (hGISA) , Mu50
(GISA), GSSA and Oxford
Staphylococcus aureus
Mu 3 (hGISA)
Mu50 (GISA)
NCTC 6571 (GSSA)
GSSA
10 9
10 8
10 7
10 6
10 5
10 4
10 3
10 2
0.5 1
2 2.5
4
8
Vancomycin concentration mg/L
GSSA < 0.9
hGISA 0.9 - 1.29
GISA  1.3
PAP-AUC
• PAP-AUC ratio
criteria:
1.5
hVISA
VISA
VSSA
1.0
0.5
0.0
0
1
2
3
Observation number
Methods of Detecting Reduced Susceptibility to
Glycopeptides in S. aureus
4
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