Related Experiment Video
Updated: Aug 2, 2026
Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds
Published on: February 14, 2018
Evaluation of broth microdilution testing parameters and agar diffusion Etest procedure for testing susceptibilities
1Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298, USA. avingrof@hsc.vcu.edu
Abstract:
The NCCLS M38-A document does not describe guidelines for testing caspofungin acetate (MK-0991) and other echinocandins against molds. This study evaluated the susceptibilities of 200 isolates of Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus to caspofungin (MICs and minimum effective concentrations [MECs]) by using standard RPMI 1640 (RPMI) and antibiotic medium 3 (M3), two inoculum sizes (10(3) and 10(4) CFU/ml), and two MIC determination criteria (complete [MICs-0] and prominent growth inhibition [MICs-2]) at 24 and 48 h. Etest MICs were also determined. In general, caspofungin MIC-2 and MEC pairs were comparable with both media and inocula (geometric mean ranges of MECs and MICs, respectively, with larger inoculum: 0.12 to 0.64 microg/ml and 0.12 to 0.44 microg/ml with RPMI versus 0.04 to 0.51 microg/ml and 0.03 to 0.21 microg/ml with M3); however, MEC results were less influenced by testing conditions than MICs, especially with the larger inoculum. Overall, the agreement between caspofungin Etest MICs and broth dilution values was higher with MECs obtained with M3 (>90%) and the large inoculum than under the other testing conditions. Because RPMI is a more stable and chemically defined medium than M3, the determination at 24 h of the easier visual MECs with RPMI and the inoculum recommended in the M38-A document appears to be a suitable procedure at present for in vitro testing of caspofungin against Aspergillus spp. Future in vitro correlations with in vivo outcome of both microdilution and Etest procedures may detect more-relevant testing conditions.
Insights
This study assessed caspofungin susceptibility in Aspergillus molds, finding RPMI medium at 24 hours with a standard inoculum is suitable for in vitro testing. This provides a reliable method for evaluating antifungal effectiveness against these fungi.
Area of Science:
- Medical Mycology
- Antifungal Susceptibility Testing
Background:
- The NCCLS M38-A guidelines lack specific methods for testing echinocandins like caspofungin against molds.
- Accurate in vitro susceptibility testing is crucial for guiding antifungal therapy.
Purpose of the Study:
- To evaluate caspofungin acetate (MK-0991) susceptibility in Aspergillus species using various testing conditions.
- To determine optimal in vitro testing parameters for caspofungin against molds.
Main Methods:
- Tested 200 Aspergillus isolates against caspofungin using RPMI 1640 and antibiotic medium 3.
- Varied inoculum sizes (10^3 and 10^4 CFU/ml) and MIC determination criteria (MICs-0, MICs-2) at 24 and 48 hours.
- Compared broth microdilution results with Etest MICs.
Main Results:
- Caspofungin MIC-2 and minimum effective concentration (MEC) pairs were comparable across media and inocula.
- MEC results showed less variability with testing conditions than MICs, particularly with larger inocula.
- Higher agreement (>90%) between Etest and broth dilution was observed with M3 and larger inoculum.
Conclusions:
- Determining MECs at 24 hours using RPMI medium and the M38-A recommended inoculum is a suitable method for in vitro caspofungin testing against Aspergillus.
- Further in vivo outcome correlations are needed to refine optimal testing conditions.
