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Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds
Published on: February 14, 2018
Comparing Etest and Broth Microdilution for Antifungal Susceptibility Testing of the Most-Relevant Pathogenic Molds
Frédéric Lamoth1, Barbara D Alexander2
1Division of Infectious Diseases and International Health, Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA Clinical Microbiology Laboratory, Department of Pathology, Duke University Medical Center, Durham, North Carolina, USA Infectious Diseases Service, Department of Medicine, Lausanne University Hospital, Lausanne, Switzerland Institute of Microbiology, Lausanne University Hospital, Lausanne, Switzerland.
Abstract:
Invasive mold infections are life-threatening diseases for which appropriate antifungal therapy is crucial. Their epidemiology is evolving, with the emergence of triazole-resistant Aspergillus spp. and multidrug-resistant non-Aspergillus molds. Despite the lack of interpretive criteria, antifungal susceptibility testing of molds may be useful in guiding antifungal therapy. The standard broth microdilution method (BMD) is demanding and requires expertise. We assessed the performance of a commercialized gradient diffusion method (Etest method) as an alternative to BMD. The MICs or minimal effective concentrations (MECs) of amphotericin B, voriconazole, posaconazole, caspofungin, and micafungin were assessed for 290 clinical isolates of the most representative pathogenic molds (154 Aspergillus and 136 non-Aspergillus isolates) with the BMD and Etest methods. Essential agreements (EAs) within ±2 dilutions of ≥90% between the two methods were considered acceptable. EAs for amphotericin B and voriconazole were >90% for most potentially susceptible species. For posaconazole, the correlation was acceptable for Mucoromycotina but Etest MIC values were consistently lower for Aspergillus spp. (EAs of <90%). Excellent EAs were found for echinocandins with highly susceptible (MECs of <0.015 μg/ml) or intrinsically resistant (MECs of >16 μg/ml) strains. However, MEC determinations lacked consistency between methods for strains exhibiting mid-range MECs for echinocandins. We concluded that the Etest method is an appropriate alternative to BMD for antifungal susceptibility testing of molds under specific circumstances, including testing with amphotericin B or triazoles for non-Aspergillus molds (Mucoromycotina and Fusarium spp.). Additional study of molecularly characterized triazole-resistant Aspergillus isolates is required to confirm the ability of the Etest method to detect voriconazole and posaconazole resistance among Aspergillus spp.
Insights
The Etest method shows promise as an alternative to broth microdilution for antifungal susceptibility testing in molds, particularly for certain non-Aspergillus species and specific antifungal drugs.
Area of Science:
- Medical Mycology
- Clinical Microbiology
- Antifungal Drug Discovery
Background:
- Invasive mold infections pose significant life-threatening risks, necessitating effective antifungal treatments.
- Emerging resistance in Aspergillus spp. and non-Aspergillus molds to existing antifungals complicates treatment strategies.
- Antifungal susceptibility testing (AST) is vital for guiding therapy, despite limited interpretive criteria for molds.
Purpose of the Study:
- To evaluate the performance of the Etest gradient diffusion method as a substitute for the standard broth microdilution (BMD) method for mold AST.
- To assess the agreement between Etest and BMD for key antifungal agents against a diverse panel of pathogenic molds.
Main Methods:
- Compared Etest and BMD for determining Minimum Inhibitory Concentrations (MICs) or Minimum Effective Concentrations (MECs) of five antifungal drugs (amphotericin B, voriconazole, posaconazole, caspofungin, micafungin).
- Tested 290 clinical mold isolates, including 154 Aspergillus and 136 non-Aspergillus species.
- Evaluated essential agreement (EA) within ±2 dilutions, with ≥90% considered acceptable.
Main Results:
- High essential agreement (>90%) between Etest and BMD was observed for amphotericin B and voriconazole against susceptible species.
- Etest showed acceptable correlation for posaconazole with Mucoromycotina but underestimated MICs for Aspergillus spp. (<90% EA).
- Excellent agreement for echinocandins was noted for highly susceptible or resistant strains, but consistency varied for intermediate strains.
Conclusions:
- The Etest method is a viable alternative to BMD for mold AST in specific scenarios, including testing amphotericin B and triazoles against non-Aspergillus molds.
- Further research is needed to validate Etest performance for detecting azole resistance in Aspergillus spp., especially with molecularly characterized isolates.
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