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

Introduction

Yeast infections, particularly those caused by Candida and Cryptococcus species, are a significant cause of morbidity and mortality, especially in immunocompromised and oncology patients. Accurate antifungal susceptibility testing (AFST) is essential for guiding appropriate therapy. The Clinical and Laboratory Standards Institute (CLSI) broth microdilution method is the reference standard for antifungal susceptibility testing of yeasts. It allows determination of minimum inhibitory concentrations (MICs) for antifungal agents under standardized laboratory conditions.

Principle

Broth Microdilution involves preparing serial two-fold dilutions of antifungal drugs in liquid medium (RPMI 1640 with MOPS buffer). Inoculum preparation: A standardized suspension of yeast cells (McFarland, diluted to the required cell density) is added to the wells of a microtiter plate containing drug dilutions. Incubation: Plates are incubated at 35 °C for 24–48 hours, depending on the yeast species. MIC determination: The MIC is the lowest drug concentration that prevents visible growth (or causes ≥50% growth reduction for certain antifungals like azoles). Controls: Growth control (no drug) and sterility control (no inoculum) are included for assay validity. The CLSI standards (e.g., CLSI M27-A4) provide detailed guidelines for reproducibility and interpretation.

Clinical Significance of Yeasts-Broth Microdilution Testing

1. Guides Antifungal Therapy – Provides reliable MIC values to help clinicians choose effective antifungal treatment.
2. Detection of Resistance – Identifies resistance in Candida auris, azole resistance in Candida glabrata, or echinocandin resistance in Candida albicans.
3. Standardization – CLSI broth microdilution is internationally recognized and ensures inter-laboratory comparability.
4. Surveillance – Used in epidemiological studies for monitoring antifungal resistance trends.
5. Quality Assurance – Reference method against which commercial or automated systems (e.g., VITEK 2, Sensititre, Etest) are validated.

Keynotes on Yeasts-Broth Microdilution Testing

• Gold standard method for yeast antifungal susceptibility testing.
• Antifungals tested: azoles (fluconazole, voriconazole, posaconazole), echinocandins (caspofungin, micafungin), polyenes (amphotericin B), and flucytosine.
• MIC endpoints vary: fungistatic vs fungicidal drugs (e.g., amphotericin B endpoint = complete inhibition; azoles = 50% inhibition).
• Clinical breakpoints (CBPs) and epidemiological cutoff values (ECVs) are used for interpretation.
• Limitations: Labor-intensive, time-consuming, requires technical expertise, and is not always feasible for routine labs.
• Alternatives: Automated systems and molecular assays, but CLSI broth microdilution remains the reference standard.

Further Reading

1. https://pmc.ncbi.nlm.nih.gov/articles/PMC7194854/
2. https://pmc.ncbi.nlm.nih.gov/articles/PMC10305799/
3. https://clsi.org/shop/standards/m27/
4. https://link.springer.com/protocol/10.1007/978-1-0716-3155-3_1
5. https://academic.oup.com/jacamr/article/4/3/dlac067/6618843
6. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/broth-dilution
7. https://pmc.ncbi.nlm.nih.gov/articles/PMC3769738/
8. https://www.nature.com/articles/s41598-024-74068-2
9. https://www.scielo.br/j/jvatitd/a/s3xnNbNzhTmCpWpSkmQfgXB/?lang=en
10. https://onlinelibrary.wiley.com/doi/full/10.1002/smtd.202100713
11. https://www.clinicalmicrobiologyandinfection.org/article/S1198-743X(14)63158-X/fulltext
12. https://www.cureus.com/articles/78906-comparison-of-clinical-laboratory-standards-institute-clsi-microdilution-method-and-vitek-2-automated-antifungal-susceptibility-system-for-the-determination-of-antifungal-susceptibility-of-candida-species.pdf
13. https://pmc.ncbi.nlm.nih.gov/articles/PMC4736391/
14. 20mL.https://www.researchgate.net/publication/244800553_Standardized_disk_diffusion_method_for_yeasts
15. http://www.mcsprogram.org/libweb/u2D9HF/243695/Antifungal%20Susceptibility%20Testing%20Clinical%20Laboratory%20And.pdf
16. https://pmc.ncbi.nlm.nih.gov/articles/PMC10305799/
17. https://www.ampath.co.za/storage/402/2017_antibiotic-guide_chapter_21.pdf