Reading_guide_BMD_v_5.0_2024_EUCAST MIC interpretation
- 1. EUCAST reading guide for broth microdilution Version 5.0 January 2024
- 2. Changes from previous version (4.0) Slide Change 17 Clarification on disregarding haze when reading MIC endpoints for cefiderocol
- 3. Broth microdilution • Broth microdilution is the reference method for antimicrobial susceptibility testing of rapidly growing aerobic bacteria, except for mecillinam and fosfomycin, where agar dilution is the reference method. • EUCAST recommends testing according to the International Standard ISO 20776-1, but with the use of MH-F broth (Mueller- Hinton broth supplemented with 5% lysed horse blood and 20 mg/L β-NAD, see instructions for preparation at www.eucast.org) for fastidious organisms. • Results are recorded as the lowest concentration of antimicrobial agent that inhibits visible growth of a microorganism, the Minimum Inhibitory Concentration (MIC), expressed in mg/L or µg/mL. 3
- 4. Reading broth microdilution Results are only valid when the following criteria are met: • Sufficient growth, i.e. obvious button or definite turbidity, in the positive growth control. • Pure culture − Check for purity by subculturing from the growth-control well immediately after inoculation onto a non-selective agar plate for simultaneous incubation. • Correct inoculum 5 x 105 CFU/mL − Viable colony counts can be performed by removing 10 µL from the growth-control well or tube immediately after inoculation and diluting in 10 mL of saline. Mix and spread 100 µL onto a non-selective agar plate. After incubation, the number of colonies should be approximately 20-80. 4
- 5. Growth appearance • Growth appears as turbidity or as a deposit of cells at the bottom of the well. The appearance of growth differs depending on the microorganism and the antimicrobial agent tested. • For round-bottom wells, growth will most often appear as a button/pellet centered in the middle. For flat-bottom wells, growth may be scattered. • Growth in antibiotic-containing wells may differ from growth seen in the positive growth control, even for pure cultures. 5
- 6. Reading MIC endpoints • Results should be read manually. The use of a mirror may facilitate reading. • If an automated reader or camera system is used, it must be calibrated to manual reading. • Read the MIC as the lowest concentration of antimicrobial agent that completely inhibits growth of the organism as detected by the unaided eye. For exceptions, see slides 12-18. 6
- 7. Trailing endpoints • Most antimicrobial agent-organism combinations give distinct endpoints. • Some agent-organism combinations may give trailing endpoints with a gradual fading of growth over 2 to 3 wells. • Unless otherwise stated, endpoints should be read at complete inhibition of growth (for exceptions, see slides 12-16). 7
- 8. Turbidity without pellet • Haze or turbidity without a pellet is often seen for Pseudomonas spp. and Acinetobacter spp. This should be regarded as growth and the endpoint read at the first well with complete inhibition (clear broth). 8
- 9. Haemolysis • For fastidious organisms tested in MH-F broth, haemolysis of the blood can be seen. This is often accompanied by turbidity or a deposit of growth (pellet). • Haemolysis with turbidity or pellet should be regarded as growth when determining endpoints. 9
- 10. Skipped wells • Occasionally a skip may be seen, i.e. a well showing no growth bordered by wells showing growth. There are several possible explanations including incorrect inoculation, contaminations, heterogenous resistance etc. • When a single skipped well occurs, retest the isolate or read the highest MIC value to avoid reporting isolates as false susceptible. • Do not report results for antimicrobial agents for which there is more than one skipped well. 10
- 11. Examples skipped wells 11 Retest or read the highest MIC value! Results invalid!
- 12. Specific reading instructions • The following antimicrobial agents require specific reading instructions: • Bacteriostatic antimicrobial agents, both with Gram- positive and Gram-negative organisms • Trimethoprim and trimethoprim-sulfamethoxazole • Cefiderocol 12
- 13. Gram-positive cocci with bacteriostatic antimicrobial agents • Disregard pinpoint growth (tiny buttons) when trailing growth occurs. 13 Doxycyline Doxycyline Fusidic acid Tetracycline
- 14. Gram-positive cocci with bacteriostatic antimicrobial agents • Disregard pinpoint growth (tiny buttons) when trailing growth occurs. 14 Linezolid Linezolid Tedizolid Tedizolid
- 15. Gram-negative organisms with bacteriostatic antimicrobial agents • Disregard pinpoint growth (tiny buttons) when trailing growth occurs. 15 Tigecycline Tigecycline Eravacycline Eravacycline
- 16. Trimethoprim and trimethoprim-sulfamethoxazole Read the MIC at the lowest concentration that inhibits ≥80 % of growth as compared to the growth control. 16 Growth control
- 17. Cefiderocol • Broth microdilution MIC determination must be performed in iron-depleted Mueller-Hinton broth and specific reading instructions must be followed. For testing conditions, see http://www.eucast.org/guidance_documents/. • The MIC is read as the first well in which the reduction of growth corresponds to a button of <1 mm or is replaced by the presence of light haze/faint turbidity (≥80% reduction in haze as compared to the growth control). • The positive control should show strong growth in the form of a button of >2 mm or heavy turbidity. • See next slide for pictures with reading examples. 17
- 18. Cefiderocol 18
- 19. 19 Interpretation of results • Make sure that MIC values for relevant Quality Control strains are within acceptable ranges before reporting results for clinical isolates. – See quality control criteria in EUCAST QC Tables (www.eucast.org). • Interpret MIC values into susceptibility categories (S, I and R) according to the current EUCAST Breakpoint Tables (www.eucast.org).