Can We Improve Antifungal Susceptibility Testing?

Abstract

Systemic antifungal agents are increasingly used for prevention or treatment of invasive fungal infections, whose prognosis remains poor. At the same time, emergence of resistant or even multi-resistant strains is of concern as the antifungal arsenal is limited. Antifungal susceptibility testing (AFST) is therefore of key importance for patient management and antifungal stewardship. Current AFST methods, including reference and commercial types, are based on growth inhibition in the presence of an antifungal, in liquid or solid media. They usually enable Minimal Inhibitory Concentrations (MIC) to be determined with direct clinical application. However, they are limited by a high turnaround time (TAT). Several innovative methods are currently under development to improve AFST. Techniques based on MALDI-TOF are promising with short TAT, but still need extensive clinical validation. Flow cytometry and computed imaging techniques detecting cellular responses to antifungal stress other than growth inhibition are also of interest. Finally, molecular detection of mutations associated with antifungal resistance is an intriguing alternative to standard AFST, already used in routine microbiology labs for detection of azole resistance in Aspergillus and even directly from samples. It is still restricted to known mutations. The development of Next Generation Sequencing (NGS) and whole-genome approaches may overcome this limitation in the near future. While promising approaches are under development, they are not perfect and the ideal AFST technique (user-friendly, reproducible, low-cost, fast and accurate) still needs to be set up routinely in clinical laboratories.

Keywords: Antifungals susceptibility testing; MALDI-TOF; computed imaging; flow cytometry; molecular biology.

Figure 1

Conventional methods currently used for AFST. (A, B) In liquid medium. (A) Broth microdilution methods in 96-well microplate: EUCAST and CLSI references and Sensititre™ Yeast One™ (B) ATB Fungus 3. (C–H) In solid agar medium. (C) Disk diffusion method. (D–H) Gradient strips diffusion method. Black arrows indicate the MIC value. (D) C albicans isolate susceptible to anidulafungin; (E) C albicans isolate resistant to fluconazole; (F) C albicans isolate susceptible to fluconazole showing a strong trailing effect (G) A fumigatus isolate susceptible to voriconazole (H) A fumigatus isolate susceptible to anidulafungin.