Rapid and Sensitive Detection of Azole-Resistant Aspergillus fumigatus by Tandem Repeat Loop-Mediated Isothermal Amplification

摘要

Invasive fungal infections caused by multiazole-resistant Aspergillus fumigatus are associated with increasing rates of mortality in susceptible patients. Current methods of diagnosing infections caused by multiazole-resistant A. fumigatus are, however, not well suited for use in clinical point-of-care testing or in the field. Loop-mediated isothermal amplification (LAMP) is a widely used method of nucleic acid amplification with rapid and easy-to-use features, making it suitable for use in different resource settings. Here, we developed a LAMP assay to detect a 34 bp tandem repeat, named TR34-LAMP. TR34 is a high-prevalence allele that, in conjunction with the L98H single-nucleotide polymorphism, is associated with the occurrence of multiazole resistance in A. fumigatus in the environment and in patients. This process was validated with both synthetic double-stranded DNA and genomic DNA prepared from azole-resistant isolates of A. fumigatus. Use of our assay resulted in rapid and specific identification of the TR34 allele with high sensitivity, detecting down to 10 genomic copies per reaction within 25 minutes. Fluorescent and colorimetric detections were used for the analysis of 11 clinical isolates as cross validation. These results show that the TR34-LAMP assay has the potential to accelerate the screening of clinical and environmental A. fumigatus to provide a rapid and accurate diagnosis of azole resistance, which current methods struggle to achieve.