BSAC Antimicrobial Susceptibility Testing & resistance Meeting, 25th October 2010 Methods of Detecting reduced susceptibility to glycopeptides in S. aureus Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus Presenter: Mandy Wootton

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 GISA/hGISA < 30 world wide None in UK UK prevalence: <1% (EARSS) 6064 isolates (1 Intermediate) MIC 0% (2001-6 BSAC Bacteraemia) 615 isolates 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 MIC <0.25 0.5 1 2 4 8 16 GSSA hGISA GISA Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

EUCAST Breakpoints GSSA hGISA GISA MIC <0.25 0.5 1 2 4 8 16 GSSA hGISA GISA Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

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 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 GRSA Detected GISA Mostly Detected 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 GSSA hGISA GISA Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

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 Disc MIC (Etest & automated) 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 Interpretative Criteria: (NOT MICs) 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 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 MHA5T Detects Most GISA MHA5T Detects Most hGISA 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 hGISA or GISA if ≥2 cols after 24hr Multi-centre study 12 labs (Europe & US) 48 strains 3 method * 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 103 / N Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus

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