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Methods of Detecting reduced susceptibility to glycopeptides in S. aureus. BSAC Antimicrobial Susceptibility Testing & resistance Meeting, 13 th May 2010. Presenter: Mandy Wootton. Phenotype. Glycopeptide Resistant Staphylococcus aureus GRSA vanA gene present, MIC VAN >3 2mg/L

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methods of detecting reduced susceptibility to glycopeptides in s aureus
Methods of Detecting reduced susceptibility to glycopeptides in S. aureus

BSAC Antimicrobial Susceptibility Testing & resistance Meeting, 13th May 2010

Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

Presenter: Mandy Wootton

phenotype
Phenotype
  • Glycopeptide Resistant Staphylococcus aureusGRSA
    • vanA gene present, MIC VAN >32mg/L
  • Glycopeptide Intermediate Staphylococcus aureusGISA
    • homogeneous resistance, mechanism unknown - MIC VAN ~4/8mg/L (Mu50)
  • heterogeneous-Glycopeptide Intermediate Staphylococcus aureushGISA
    • 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
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

GSSA

hGISA

GISA

BSAC Breakpoints

MIC <0.25 0.5 1 2 4 8 16

Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

eucast breakpoints

MIC <0.25 0.5 1 2 4 8 16

GSSA

hGISA

GISA

EUCAST Breakpoints

Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

disc diffusion 1
Disc Diffusion 1
  • ISA with 1:10 of 0.5 McFarland inoculum
  • 5g Vancomycin disc
  • Incubation at 35-37°C for 18-20h
  • Interpretive Criteria:
    • Resistance - 14mm
    • Susceptible - 15mm
  • GRSA: Detected
  • GISA: NOT Reliably Detected
  • hGISA: NOT Detected

Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

disc diffusion 2
Disc Diffusion 2
  • 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 18-20h
  • Separation of phenotypes
  • 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

mic determination 1

GSSA

hGISA

GISA

MIC Determination 1
  • Standard Etest
    • 0.5 McFarland on ISA, 18-20h
    • BPs 2 (S)/ >2 (I/R)

GRSA Detected

GISA Mostly Detected

hGISA mostly missed

MIC <0.25 0.5 1 2 4 8 16

Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

mic determination 2
MIC Determination 2

Automated (PHX)

  • Repeat 1 MRSA/GSSA x22
  • GSSA x10

hGISA x10

GISA x10

Some GISA & hGISA identified

Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

summary
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
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
GRD Etest
  • 0.5 McFarland inoculum
  • Mueller Hinton + blood
  • 48h incubation at 35-37°C in air
    • Reading at 24h and 48h
  • Interpretive criteria:

GISA or hGISA if VA or TP 8mg/L

    • GISA if GRD+ and standard VA MIC 4mg/L
    • hGISA if GRD+ and standard VA MIC <4mg/L

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
Screening Agar 1
  • Multi-centre study
    • 12 labs (Europe & US)
    • 48 strains
    • 3 method
  • Mueller Hinton Agar + 5mg/L TP (MHA5T)
    • 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

* 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
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 103 / N

Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

pap auc 2
PAP-AUC 2
  • Log viable count vs. VA concn graph
  • Ratio = AUC of test isolate/AUC of Mu3
  • PAP-AUC ratio criteria:

GSSA < 0.9

hGISA 0.9 - 1.29

GISA  1.3

Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

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