1. 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, 13 th May 2010 Presenter: Mandy Wootton

2. Methods of Detecting Reduced Susceptibility to 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

3. 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

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

5. 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

6. 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

7. 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 conc n discs of V and T differentiate GISA & GSSA None accurate for ID of hGISA

8. Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus MIC Determination 1 Standard Etest –0.5 McFarland on ISA, 18-20h –BPs 2 (S)/ >2 (I/R) MIC <0.25 0.5 1 2 4 8 16 GSSA hGISA GISA GRSA Detected GISA Mostly Detected hGISA mostly missed

9. 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

10. 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

11. 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

12. 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 1)GISA if GRD+ and standard VA MIC 4mg/L 2)hGISA if GRD+ and standard VA MIC <4mg/L Detects all GISA, majority at 24h Detects most hGISA and GISA 2% false positives

13. Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus Screening Agar 1 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 Multi-centre study –12 labs (Europe & US) –48 strains –3 method MHA5T Detects Most GISA MHA5T Detects Most hGISA Up to 24% false positives * MHA5T: false positives reduced when  2 cols at 48h used

14. 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 10 6 / 10 3 10 6 / 10 3 10 6 / 10 3 10 3 / N 10 3 / N 10 3 / N

15. Methods of Detecting Reduced Susceptibility to Glycopeptides in S. aureus PAP-AUC 2 Log viable count vs. VA conc n graph Ratio = AUC of test isolate/AUC of Mu3 PAP-AUC ratio criteria: GSSA< 0.9 hGISA0.9 - 1.29 GISA 1.3