Methodological problems for the detection of resistant S. aureus: - PowerPoint PPT Presentation

Methodological problems for the detection of resistant S. aureus: MRSA, VISA Y. Glupczynski Laboratoire de bactériologie Cliniques Universitaires UCL de Mont-Godinne Université Catholique de Louvain Symposium on Staphylococcus aureus (Bruxelles 11/01/2007)

Methicillin-resistance in S. aureus 1. Acquisition of exogeneous PBP2a ( mecA gene) � Cf SCC mec ⇒ Transcription of mecA ⇒ synthesis of PBP 2a (transpeptidases with decreased affinity to penicillins, cephalosporins and carbapenems- 2. Non mec A mediated resistance (rare) � Hyperproduction of β -lactamases (plasmid) � Production of meticillinases (plasmid) � Hyperproduction of normal PBPs (BORSA) Modification of endogeneous PBPs (1,2&4) (MODSA)

Staphylococcal Cassette Chromosome mec (SCC mec ) mec complex (A, B, C) ccr complex (A/ B) SCC mec I SCC mec I I SCC mec I I I 5 different types SCC mec I V based on polymorphismi in conserved genes SCC mec V pT181 mecA mecA on mobile genetic element (21-67 kb) orfX pUB110 may contain other resistance genes IS 431 Tn 554

Heterogeneous expression of methicillin resistance Hetero-resistant strains Homo-resistant strains 10 -6 resistant subpopulation Tomasz A. et al. J Clin Microbiol. 1991; 35:124

Facteurs influencing transcription of mecA • [NaCl] ↑ Auxiliary genes ( fem, fmt, sarA, agr… ) •T° •Osmolality •pH Level of methicillin (Oxa) resistance blaZ penicillinase regulatory genes mecA regulatory genes ( blaI , blaR1 ) ( mecI , mecR1 )

Methods for detection of MRSA • Oxacillin disk test (1µg, 5 µg) • Oxacillin agar screen • Cefoxitin disk test • Automated systems (Vitek2, Phoenix,…) • MIC (agar, microbroth, E-test) • MRSA screen (PBP2a latex) • Detection of mecA gene

Accuracy & TAT of Culture-Based MRSA Detection Tests Method Sensitivity Specificity TAT Cefoxitin disk diffusion 97-98 >97 18h Oxacillin agar screen 95-98 >95 24h Broth microdilution >98 >99 24h Microscan 93-97 >86 8h Vitek 2 88-100 >99 6-12h Phoenix BD 98-100 >99 6-12h PBP2a latex 97-100 >97 15 min Swenson JCM 2001;39:3785 Felten JCM 2002;40:2766 Nonhoff 14th ECCMID 2004 Flayhart JCM 2005; 43:5536 Brown JAC 2005;56:1000.

Sensitivities of various methods for detection of 83 MRSA clinical isolates Felten A. et al. JCM 2002 40:2766

Cefoxitin (30 µg) zone diameters • More potent inducer of the mecA regulatory genes S. aureus 160 mecA + 140 CLSI 2007 mecA - • Easier to read (18 h instead of 24h) 120 100 80 60 • Disk diffusion breakpoints 40 20 – Susceptible > 20 mm for S. aureus 0 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 >28 – Susceptible > 25 mm for CNS CoNS 80 mecA + mecA - 60 Organism Oxacillin Cefoxitin 40 Sens Spec Sens Spec 20 S. aureus 86-98 74-99 98-100 100 0 CoNS 94-99 79-89 90-99 96-97 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 >28 Swenson JM et al. JCM 2005 43:3818

Disk diffusion test for prediction of mecA- mediated resistance in Staphylococci (CLSI M100-S17) S. aureus and S. lugdunensis Zone diameter (mm) S I R ≤ 10 ≥ 13 Oxacillin 11-12 ≤ 21 ≥ 22 Cefoxitin -

Sensitivities of disk diffusion, oxascreen and automated systems for detection of MRSA All MRSA Hetero-MRSA (n = 26) Homo-MRSA (n = 72) 100 75 Percent 50 25 0 Oxascreen DD Cefox DD oxa Vitek 2 BD Phoenix BD Phoenix oxa cefox Nonhoff C. et al. 14 th ECCMID 2004

Belgian national external quality control 193 participating labs One isolate hetero-resistant to oxacillin – mecA positive with MIC to oxacillin of 4 µg/ml – Susceptible to quinolones, MLS and aminoglycosides – Resistant to fusidic acid Only 82% reported as MRSA 60 N of labs responding 50 40 Resistant Susceptible 30 20 10 0 Disk oxacillin Disk cefoxitin Vitek 2 BD Phoenix ATB

Most prevalence CA-MRSA clone in Belgium (ST80 SCC mec IV) P: Penicilline G P OX FOX Va OX: oxacilline Fox: cefoxitine Va: vancomycine L: lincomycine E: érythromycine E L Pt TET Pt: pristinamycine Tet: tétracycline FA: ac. fusidique C: chloramphénicol OFX: ofloxacine OFX FA C Sxt Sxt: cotrimoxazole Ft: furanes RA: rifampicine TM: tobramycine TM GM: gentamicine GM Ft RA

Reduced susceptibility to glycopeptides: VISA - VRSA

Glycopeptide cut-off values MI C for vancomycin MI C for teicoplanin (µg/ ml) (µg/ ml) S I R S I R ≤ 2 ≥ 32 ≤ 8 ≥ 32 CLSI 8-16 16 ≤ 4 ≥ 32 ≤ 4 ≥ 32 SFM 8-16 8-16 ≤ 4 > 4 BSAC

hVISA - VISA • With the current CLSI breakpoints, no differentiation between VISA and hVISA • What about strain with MIC between 2 and 4 µg/ml (E-test) ? NB: Etest not recommended by CLSI !

Current methods for determination of susceptibility to glycopeptides • Disk diffusion • Agar screening – BHI agar + (4) or 6 µg/ml Vanco (CLSI) – MH agar + 5 µg/ml Teico (SFM, EARSS) • Automated systems • MIC determination – Agar – Broth microdilution (CLSI) – E-test

Pittfalls and problems in detection of glycopeptide resistance in Staphylococci • Poor diffusion in agar (disk diffusion) • Expression of resistance slow /low (rapid automate systems !) • High inoculum effect (teicoplanin) • Type of medium (Brand/batch) (teicoplanin) • No distinction in MIC distribution between GSSA and certain GISA isolates (hGISA) • No molecular reference tests for categorization of GISA (population analysis profiles)

Comparative MIC values of 294 putative hGISA/GISA strains Screening by growth on BHIA +4 µg teico, Macromethod E test and PAP 11% hGISA Garnier et al. JAC 2006

Screening tests for GISA (I) • Vancomycin agar screen test (CLSI ) – 10 µl of 0.5 MF suspension on BHI agar + 6 µg vanco – Incubation: 35°C / ambiant air / 24 h – Test positive: if ≥ 2 colonies • Teicoplanin agar screen test (SFM) – 10 µl of 2 MF suspension on MH agar + 5 µg teico – Incubation: 35°C / ambiant air / 24-48 h – Test positive: if ≥ 4 colonies Positive test = presumed reduced susceptibility !

Screening tests for GISA (II) • Modified E-test (Macromethod) – 2 MF suspension in MH broth; 200 µl plated on BHI agar – Incubation: 35°C / ambiant air / 48 h – Test positive: MIC vancomycin AND teicoplanin ≥ 8 µg/ml or MIC teicoplanin ≥ 12 µg/ml Do not round up MIC value (ie: 6 µg/ml -> 8 µg/ml !) Positive test = presumed reduced susceptibility ! Walsh et al. JCM 2001

Glycopeptide E-test MICs Macromethod (2 McF / BHI / 48 h) HIP5827 (GISA) ATCC29213 (Peni-S MSSA) Vanco MIC 2 µg/ml Vanco MIC 16 µg/ml Teico MIC 64 µg/ml Teico MIC 3 µg/ml Slide kindly provided by O. Denis

Performance of three screening methods for detecting GISA isolates Multicentric study (12 labs, Europe - USA- Australia) 48 strains (15 GISA, 15 hGISA, 15 GSSA, 3 control strains) Screening Sensitivity Specificity PPV NPV method (% ) (% ) (% ) (% ) BHIA6V 35.2 97.4 98.4 45.2 MHA5T 85.9 75.5 82.2 79.1 Macro ET 82.0 89.1 94.0 74.4 Wootton et al. JCM 2006

Algorithm for the detection of GISA/hGISA S. aureus strains Positive Agar Screening test Treatment failure with (Vanco or Teico) glycopeptide or MIC of Vanco or teico ≥ 4 µg/ml Macromethod E-test (BHIA, 2 MF, 48 h) Positive Negative Vanco and teico ≥ 8 µg/ml or Teico ≥ 12 µg/ml Confirmation by Susceptible Population analysis (PAP) + MIC (MH, 0.5 McF, 24 h) GISA Hetero-GISA Howden et al. EJCMID 2005 Denis et al. NosoInfo 2006

Confirmatory tests for GISA • MI C determination – Microbroth dilution (CLSI), Agar dilution, E-test – Medium: Mueller-Hinton – Inoculum: 0.5 McF – Incubation: 35°C / ambiant air / 24 h • Population analysis studies – Reference laboratory

Glycopeptide E-test MICs CLSI method (0,5 McF / MH / 24 h) HIP5827 (GISA) ATCC29213 (Peni-S MSSA) Vanco MIC 0.75 µg/ml Vanco MIC 8 µg/ml Teico MIC 16 µg/ml Teico MIC 1 µg/ml Slide kindly provided by O. Denis

Population analysis of VSSA, hVISA, VISA and VRSA isolates 8 VRSA 7 6 Log CFU/ml 5 VI SA 4 hVI SA 3 VSSA 2 1 0 1 2 3 4 5 6 7 8 9 10 11 12 Vancomycin concentration (µg/ml) Adapted from Liu c. et al. 2003. Antimicrob Agents Chemother. 47:3040

Vancomycin-resistant MRSA isolate VRSA Teico MIC Van MIC Slide kindly provided by 24 µg/ml >256 µg/ml O. Denis

Automated methods for determination of vancomycin MIC (µg/ml) with VRSA Microscan Vitek Vitek2 Michigan > 16 > 32 8* Pennsylvania 2-4 2 2* * * true MIC of 1024 µg/ml * * true MIC of 32 µg/ml Automated systems fails to detect VRSA ! Cheng et al. NEJM 2003; 348: 1342-7 Whitener SHEA 2003

Unacceptable methods • Automated methods , e.g. VITEK, Phoenix, Microscan: did not identify accurately Hershey & NYC VRSAs with MICs of 32-64 µg/ml • Disk diffusion alone (for VRSA ?) • LABORATORIES USING THE ABOVE MUST ADD A VANCO (6 µg/ml) or a TEICO (5 µg/ml) AGAR SCREEN PLATE

Acceptable methodology Non automated methodology • NCCLS broth microdilution • Agar dilution • E test (0.5 Mac Farland, 24h incubation)