Rapid antifungal susceptibility testing for Aspergillus fumigatus using a loop-mediated isothermal amplification method

Abstract

In patients with hematological diseases and immunodeficiency, aspergillosis is associated with high mortality, and the early selection of effective antifungal agents is critical. Considering the emergence of azole-resistant Aspergillus and the skill- and time-intensive nature of Clinical and Laboratory Standards Institute (CLSI)-compliant broth microdilution (BMD), there is an urgent need for a rapid and simple antifungal susceptibility testing method. Here, we adapted a loop-mediated isothermal amplification (LAMP)-based phenotypic approach to detect azole resistance in Aspergillus fumigatus, using itraconazole (ITCZ) as the index drug. We evaluated 26 isolates (20 ITCZ-susceptible and 6 ITCZ-resistant strains), whose reference minimum inhibitory concentrations (MICs) were determined using CLSI M38 BMD method. The fungi were incubated for 24 h in Roswell Park Memorial Institute medium (RPMI) medium with and without ITCZ (4 μg/mL), followed by LAMP amplification with real-time monitoring. The time to positivity was recorded as the threshold time (Tt), and susceptibility is summarized as ΔTt = Tt (+ITCZ) - Tt (-ITCZ). The Tt values showed a strong negative correlation with fungal suspension concentration (Spearman r = -0.9686, p < 0.0001). Among the clinical and reference isolates, the ΔTt values ranged from +200 to +592 s for susceptible strains and - 14 to +140 s for resistant strains. A receiver operation characteristic (ROC) curve-derived ΔTt cutoff of 150 s perfectly discriminated all isolates (sensitivity 100% [95% confidence interval (CI), 54.1-100.0]; specificity 100% [95% CI, 83.2-100.0]; AUC 1.00). Our approach produced categorical calls after 24 h of incubation and approximately 1 h of assay time, substantially faster than standard BMD. A rapid and simple LAMP-based antifungal susceptibility approach using ΔTt after brief drug exposure accurately identified ITCZ-resistant A. fumigatus.

Keywords: Antimicrobial resistance; Antimicrobial susceptibility testing; Aspergillus fumigatus; Loop-mediated isothermal amplification.