Introduction
Table of Contents
Invasive mold infections such as those caused by Aspergillus, Fusarium, Scedosporium, and Mucorales present significant clinical challenges, especially in immunocompromised hosts. Mortality rates are high, and empirical antifungal therapy often fails due to intrinsic or acquired resistance. Standardized antifungal susceptibility testing (AFST) is therefore essential. The Clinical and Laboratory Standards Institute (CLSI) provides a reference broth microdilution method (document M38, currently in its 3rd edition) specifically for filamentous fungi (molds). This method allows accurate determination of minimum inhibitory concentrations (MICs) for antifungal agents, guiding clinical therapy, epidemiological surveillance, and resistance detection.
Principle
- Microdilution Format: Antifungal agents are serially diluted two-fold in RPMI 1640 medium buffered with MOPS in sterile microtiter plates.
- Inoculum Standardization: Mold conidia (or hyphal fragments where spores are not produced) are adjusted to a standard inoculum density (usually 0.4–5 × 10⁴ CFU/mL).
- Incubation: Plates are incubated at 35 ± 2 °C for 24–48 hours, though some slow-growing molds may require up to 72–96 hours.
- Endpoint Determination:
- For azoles and echinocandins → MIC is the lowest concentration showing significant growth reduction (typically ≥50%).
- For amphotericin B, → MIC is the lowest concentration showing complete growth inhibition.
- Quality Control (QC): Reference strains such as Aspergillus fumigatus ATCC 204305 and Candida parapsilosis ATCC 22019 are included to ensure reproducibility.
Clinical Significance
- Therapeutic Guidance: Provides MIC values for molds against triazoles, amphotericin B, echinocandins, and newer antifungals.
- Resistance Detection: Identifies azole resistance in Aspergillus fumigatus (CYP51A mutations), amphotericin B resistance in Fusarium, and echinocandin resistance in rare molds.
- Epidemiological Data: Supports global surveillance of antifungal susceptibility trends, important for outbreak investigations and guideline development.
- Comparison with Commercial Methods: CLSI broth microdilution remains the gold standard against which commercial systems (e.g., Sensititre YeastOne, VITEK MS) are validated.
- Patient Outcomes: Ensures optimized antifungal therapy, especially in high-risk hematology, oncology, and transplant patients.
Keynotes
- CLSI M38 broth microdilution is the reference standard for antifungal susceptibility testing of filamentous fungi.
- It provides reproducible MICs that correlate with clinical response when interpreted with epidemiological cutoff values (ECOFFs).
- Requires pure, viable isolates and precise inoculum preparation for accuracy.
- Interpretation breakpoints are limited for some molds, but ECOFFs help distinguish wild-type from resistant strains.
- Despite being time- and labor-intensive, it is critical for resistance monitoring and validating rapid diagnostic platforms.
- Plays a vital role in guiding personalized antifungal therapy and preventing treatment failures.
Further Readings
- https://clsi.org/shop/standards/m38/
- https://www.amhsr.org/articles/pattern-of-antifungal-susceptibility-in-pathogenic-molds-by-microdilution-method-at-a-tertiary-care-hospital-4370.html
- https://www.clinicalmicrobiologyandinfection.org/article/S1198-743X(14)62817-2/pdf
- https://www.researchgate.net/publication/7557533_Quality_Control_and_Reference_Guidelines_for_CLSI_Broth_Microdilution_Susceptibility_Method_M38-A_Document_for_Amphotericin_B_Itraconazole_Posaconazole_and_Voriconazole
- https://pmc.ncbi.nlm.nih.gov/articles/PMC10305799/
- https://www.ncbi.nlm.nih.gov/books/NBK544375/
- https://www.clinicalmicrobiologyandinfection.org/article/S1198-743X(14)62817-2/fulltext
- https://scholars.uthscsa.edu/en/publications/antifungal-susceptibility-of-yeasts-and-filamentous-fungi-by-clsi
- https://pmc.ncbi.nlm.nih.gov/articles/PMC7194854/
- https://pmc.ncbi.nlm.nih.gov/articles/PMC1932988/
- https://pmc.ncbi.nlm.nih.gov/articles/PMC4572526/