TABLE 6.

Methodology for screening and confirmation of hVISA and VISA strainsb

MethodInoculumInterpretationDescriptionReference(s) or source
Vancomycin MICPrepare a 0.5-McFarland-standard direct-colony suspension in water, saline, or MHB, dilute, and inoculate cation-adjusted MHB to final concn of 5 × 105 CFU per ml; incubate a full 24 h at 35°C ± 2°CRead with unaided eye and determine lowest concn with complete inhibition of growthInoculum prepared from overnight growth on nonselective agar; control strain: ATCC 29213 (S. aureus)53
MHA + teicoplanin at 5 μg per ml (MHA5T)Prepare a 2-McFarland-standard direct-colony suspension; 10 μl onto plate; incubate for 48 h at 35°CaAny growth positiveInoculum prepared from growth on blood agar plate overnight389, 393
Macromethod Etest (MET)Pipette 200 μl of a 2-McFarland-standard suspension onto a 90-mm BHI plate and swab evenly; dry the agar surface (15 to 20 min) and then apply Etest strips; incubate for 48 h at 35°CUse oblique light and magnifying glass or plate microscope to read point of complete inhibition (Fig. 5); positive result, vancomycin MIC of ≥8 and teicoplanin MIC of ≥8 μg per ml or teicoplanin MIC of ≥12 μg per mlInoculum prepared from overnight growth on blood agar plate; beware of adherent growth if plate not dry; do not convert a result of 6 μg per ml to the next upper dilution; control strains: ATCC 29213 (S. aureus), ATCC 700699 (Mu50), ATCC 29212 (Enterococcus faecalis)EAS 003 product information; AB Biodisc, Solna, Sweden
Etest GRDUsing sterile cotton swab, inoculate MHA with 5% sheep or horse blood from a 0.5-McFarland-standard suspension in MHB; after dipping cotton swab into broth, press against side of tube to remove excess fluid; dry the agar surface (15 to 20 min), and then apply Etest GRD strip; read after 24 and 48 h at 35°CVancomycin or teicoplanin MIC of ≥8 μg per mlInoculum prepared from overnight growth on blood agar plate; control strains: ATCC 29213 (S. aureus), ATCC 700699 (Mu50), ATCC 700698 (Mu3)Product information, AB Biodisc, Solna, Sweden
PAP/AUCCulture grown overnight in TSB diluted to 10−3 and 10−6; inoculate BHIA with vancomycin at 0, 0.5, 1, 2, 2.5, and 4 μg per ml using a spiral plater; incubate for 48 h prior to counting coloniesPlot graph of CFU per ml vs vancomycin concn and calculate AUC; determine ratio of AUC of test organism vs Mu3 (ATCC 700698); if AUC ratio is ≥0.9 and vancomycin MIC is ≤2 μg per ml, the isolate is hVISAMu3 should be run in parallel with test organism; software such as GraphPad Prism (San Diego, CA) is available for calculating AUC385, 387
  • a Note that the European Antimicrobial Resistance Surveillance Scheme suggests an inoculum using 10 μl of a stationary-phase culture grown overnight in BHIB (http://www.rivm.nl/earss/).

  • b TSB, tryptone soy broth.