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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

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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

hGISA0.9 - 1.29

GISA 1.3

Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus


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