Yeasts-Broth Microdilution Testing (EUCAST): Introduction, Principle, Clinical Significance, and Keynotes

Introduction

Fungal infections caused by yeasts, particularly Candida and Cryptococcus species, represent a significant clinical challenge in immunocompromised and critically ill patients. Antifungal susceptibility testing (AFST) is crucial for guiding therapy, particularly in cases of treatment failure or the emergence of resistance. The broth microdilution method is a standardized method by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). It is a reference technique designed to provide reproducible and clinically relevant minimum inhibitory concentration (MIC) values for antifungal agents. Compared to empirical therapy, this method ensures optimized patient outcomes through evidence-based drug selection.

Principle

Microdilution format: Serial two-fold dilutions of antifungal drugs are prepared in RPMI 1640 medium buffered with MOPS, dispensed into microtiter plates. Inoculum preparation: Standardized yeast suspensions (usually 0.5–2.5 × 10⁵ CFU/mL) are inoculated into each well. Incubation: Plates are incubated at 35 ± 2 °C, generally for 24 hours (sometimes extended to 48 hours for slow-growing yeasts). Endpoint reading: MIC is defined as the lowest antifungal concentration that causes a significant reduction (≥50% for azoles/echinocandins; complete inhibition for amphotericin B) in visible growth compared to the drug-free control. Quality control: Reference strains (Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258) are tested alongside clinical isolates to ensure accuracy.

Clinical Significance

1. Guides therapy: Provides clinicians with MIC values that inform the choice of azoles, echinocandins, amphotericin B, or flucytosine.
2. Resistance detection: Detects emerging resistance, e.g., fluconazole resistance in Candida albicans or echinocandin resistance in Candida glabrata.
3. Standardization: EUCAST ensures harmonized breakpoints and epidemiological cutoff values (ECOFFs) across Europe and globally.
4. Improved outcomes: Evidence-based antifungal selection reduces morbidity, mortality, and healthcare costs in invasive fungal infections.
5. Research utility: Supports surveillance programs, clinical trials, and studies on resistance mechanisms.

Keynotes

1. EUCAST broth microdilution is the gold standard reference method for yeast antifungal susceptibility testing in Europe.
2. Provides quantitative MIC values with high reproducibility.
3. Requires standardized inoculum, medium, and incubation to minimize variability.
4. Interpretation is based on EUCAST clinical breakpoints and ECOFFs.
5. Plays a crucial role in detecting antifungal resistance and guiding therapy in invasive fungal infections.
6. Despite being labor-intensive, it remains the benchmark against which commercial systems (e.g., VITEK 2, Sensititre YeastOne) are validated..

Further Readings

• https://www.eucast.org/ast_of_bacteria
• https://www.eucast.org/eucast_news/news_singleview?tx_ttnews%5Btt_news%5D=129&cHash=da0d8ce81cacbca2734ecfde6dd5385d
• https://www.eucast.org/ast_of_bacteria/mic_testing_services_from_eucast
• https://www.eucast.org/ast_of_bacteria/mic_determination
• https://www.clinicalmicrobiologyandinfection.org/article/S1198-743X(14)62817-2/pdf
• https://www.eucast.org/ast_of_bacteria/disk_diffusion_methodology
• https://pmc.ncbi.nlm.nih.gov/articles/PMC7913839/