Manual & Automated

Antifungal susceptibility testing (AFST) is essential for guiding appropriate antifungal therapy, especially in the face of increasing antifungal resistance. We’ll look into both manual and automated methods, covering their principles, procedures, advantages, and limitations

Antifungal Susceptibility Testing (AFST)

Purpose: To determine the susceptibility of a fungal isolate to various antifungal agents
Importance:
- Guides appropriate antifungal therapy
- Detects antifungal resistance
- Monitors trends in antifungal resistance
- Helps optimize antifungal dosing
Standard Methods: Standardized methods are essential for ensuring accurate and reproducible results. The Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) provide guidelines for AFST

Manual Methods

Broth Microdilution (BMD)
- Principle: A series of two-fold dilutions of antifungal agents are prepared in a broth medium in microtiter plates. A standardized inoculum of the fungal isolate is added to each well, and the plates are incubated for a specified time. The minimum inhibitory concentration (MIC) is determined as the lowest concentration of the antifungal agent that inhibits visible growth of the fungus
- Procedure
  1. Inoculum Preparation: Prepare a standardized inoculum of the fungal isolate in a suitable broth medium (e.g., RPMI 1640)
  2. Antifungal Dilutions: Prepare two-fold dilutions of the antifungal agents in the same broth medium
  3. Microtiter Plate Setup: Dispense the antifungal dilutions into the wells of a microtiter plate
  4. Inoculation: Add the standardized inoculum to each well
  5. Incubation: Incubate the microtiter plate at the appropriate temperature (e.g., 35°C for yeasts, 25-30°C for molds) for the specified time (e.g., 24-48 hours for yeasts, up to 72 hours for molds)
  6. Reading: Examine the wells for visible growth and determine the MIC as the lowest concentration of the antifungal agent that inhibits visible growth
- Advantages
  - Gold standard method
  - Provides quantitative MIC values
  - Can be used for a wide range of fungi and antifungal agents
  - Relatively inexpensive
- Limitations
  - Labor-intensive
  - Time-consuming
  - Subjective interpretation
  - Requires technical expertise
  - Prone to errors
Etest
- Principle: A plastic strip containing a gradient of antifungal concentrations is placed on an agar plate inoculated with the fungal isolate. The antifungal agent diffuses into the agar, creating a concentration gradient. After incubation, an elliptical zone of growth inhibition is observed, and the MIC is determined as the concentration at which the zone edge intersects the strip
- Procedure
  1. Inoculum Preparation: Prepare a standardized inoculum of the fungal isolate in a suitable broth medium
  2. Agar Plate Inoculation: Swab the inoculum onto the surface of an agar plate (e.g., Mueller-Hinton agar with glucose and methylene blue for yeasts, RPMI agar for molds)
  3. Etest Strip Application: Apply the Etest strip to the agar surface
  4. Incubation: Incubate the agar plate at the appropriate temperature for the specified time
  5. Reading: Examine the plate for the elliptical zone of growth inhibition and determine the MIC as the concentration at which the zone edge intersects the strip
- Advantages
  - Easy to perform
  - Provides quantitative MIC values
  - Can be used for a wide range of fungi and antifungal agents
  - Can be used for fastidious organisms
- Limitations
  - More expensive than broth microdilution
  - Can be difficult to read for some fungi
  - May not be as accurate as broth microdilution for some antifungal agents

Automated Methods

Principle: Automated systems streamline AFST by automating various steps, such as inoculation, incubation, reading, and interpretation of results. They often use pre-packaged test kits and software to analyze data
Common Automated Systems
- Vitek 2 Compact (bioMérieux): Uses pre-filled cards containing various antifungal agents to determine the susceptibility of yeasts and molds based on their growth patterns
- MicroScan (Beckman Coulter): Employs microdilution panels containing various antifungal agents to determine the MICs of antifungals for yeasts and molds
- Sensititre (Thermo Fisher Scientific): Uses microdilution plates containing various antifungal agents to determine the MICs of antifungals for yeasts and molds
Advantages
- Faster turnaround time
- Increased throughput
- Reduced labor costs
- Improved standardization
- Objective interpretation of results
Limitations
- Higher initial cost
- Limited species coverage
- May not be suitable for all fungal species
- Requires maintenance and calibration
- Can be prone to errors if not properly validated
Procedure (General)
1. Inoculum Preparation: Prepare a standardized inoculum of the fungal isolate
2. Test Setup: Load the inoculum and the appropriate test kit (card or panel) into the automated system
3. Incubation and Reading: The system automatically incubates the test and reads the results at specified intervals
4. Interpretation: The system analyzes the growth patterns and determines the MICs or categorical interpretations (susceptible, intermediate, resistant)
Considerations
- Follow the manufacturer’s instructions carefully
- Ensure proper maintenance and calibration of the system
- Validate the system for use with specific fungal species and antifungal agents
- Be aware of the limitations of the system and confirm results with manual methods if necessary

Key Takeaways

Antifungal susceptibility testing (AFST) is essential for guiding appropriate antifungal therapy
Manual methods (broth microdilution and Etest) are reliable but labor-intensive
Automated methods streamline AFST and improve turnaround time but have higher initial costs
The choice of AFST method depends on the laboratory’s resources, the types of fungi encountered, and the clinical needs

Key Terms

Antifungal Susceptibility Testing (AFST): A laboratory test to determine the susceptibility of a fungal isolate to various antifungal agents
Minimum Inhibitory Concentration (MIC): The lowest concentration of an antifungal agent that inhibits the visible growth of a fungus
Broth Microdilution (BMD): A manual method for AFST that involves preparing two-fold dilutions of antifungal agents in a broth medium
Etest: A manual method for AFST that uses a plastic strip containing a gradient of antifungal concentrations
Automated System: A laboratory instrument that automates various steps of AFST
CLSI (Clinical and Laboratory Standards Institute): A non-profit organization that develops standards for laboratory testing
EUCAST (European Committee on Antimicrobial Susceptibility Testing): A European organization that develops standards for antimicrobial susceptibility testing
RPMI 1640: A common broth medium used for AFST
Mueller-Hinton Agar: A common agar medium used for bacterial susceptibility testing that is sometimes supplemented with glucose and methylene blue for yeast susceptibility testing
Categorical Interpretation: The classification of a fungal isolate as susceptible, intermediate, or resistant to an antifungal agent based on the MIC value
Quality Control (QC): Procedures used to monitor the accuracy and precision of laboratory tests
Breakpoint: The MIC value that separates susceptible isolates from resistant isolates
Antifungal Stewardship: A program to promote the appropriate use of antifungal agents
Mould-Specific Identification: Refers to laboratory techniques, including microscopic examination and molecular assays, used to accurately identify specific mold species in clinical samples, aiding in diagnosis and treatment of infections caused by these fungi
Panfungal PCR: is a molecular diagnostic technique that utilizes polymerase chain reaction (PCR) to amplify and detect conserved DNA sequences present in a wide range of fungi, allowing for the identification of fungal infections, even when traditional culture methods are negative or impractical

Antifungal Susceptibility

Antifungal Classes