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

Area of Science:

• Mycology
• Infectious Diseases
• Molecular Biology

Background:

• Aspergillosis poses a high mortality risk in immunocompromised patients.
• Emergence of azole-resistant Aspergillus strains necessitates rapid susceptibility testing.
• Current Clinical and Laboratory Standards Institute (CLSI)-compliant broth microdilution (BMD) is time-consuming and labor-intensive.

Purpose of the Study:

• To develop and evaluate a rapid, simple loop-mediated isothermal amplification (LAMP)-based phenotypic method for detecting itraconazole (ITCZ) resistance in Aspergillus fumigatus.
• To compare the LAMP method's performance against CLSI M38 BMD reference method.

Main Methods:

• Adapted a LAMP assay for phenotypic antifungal susceptibility testing using ITCZ.
• Incubated 26 Aspergillus isolates (20 susceptible, 6 resistant) with/without ITCZ for 24 hours.
• Measured time to positivity (Tt) and calculated ΔTt = Tt (+ITCZ) - Tt (-ITCZ) for real-time LAMP monitoring.

Main Results:

• A strong negative correlation was observed between Tt values and fungal suspension concentration (Spearman r = -0.9686, p < 0.0001).
• ΔTt values clearly distinguished susceptible (+200 to +592s) from resistant (-14 to +140s) strains.
• A ΔTt cutoff of 150s achieved 100% sensitivity and 100% specificity (AUC 1.00).

Conclusions:

• The developed LAMP-based method provides accurate and rapid antifungal susceptibility results for ITCZ-resistant Aspergillus fumigatus.
• This approach significantly reduces testing time (24h incubation + 1h assay) compared to standard BMD.
• The method offers a practical alternative for early selection of effective antifungal agents in clinical settings.