Rapid Detection of Azole-Resistant Aspergillus fumigatus in Clinical and Environmental Isolates by Use of a Lab-on-a-Chip Diagnostic System

Abstract

Aspergillus fumigatus has widely evolved resistance to the most commonly used class of antifungal chemicals, the azoles. Current methods for identifying azole resistance are time-consuming and depend on specialized laboratories. There is an urgent need for rapid detection of these emerging pathogens at point-of-care to provide the appropriate treatment in the clinic and to improve management of environmental reservoirs to mitigate the spread of antifungal resistance. Our study demonstrates the rapid and portable detection of the two most relevant genetic markers linked to azole resistance, the mutations TR34 and TR46, found in the promoter region of the gene encoding the azole target, cyp51A. We developed a lab-on-a-chip platform consisting of: (1) tandem-repeat loop-mediated isothermal amplification, (2) state-of-the-art complementary metal-oxide-semiconductor microchip technology for nucleic-acid amplification detection and, (3) and a smartphone application for data acquisition, visualization and cloud connectivity. Specific and sensitive detection was validated with isolates from clinical and environmental samples from 6 countries across 5 continents, showing a lower limit-of-detection of 10 genomic copies per reaction in less than 30 minutes. When fully integrated with a sample preparation module, this diagnostic system will enable the detection of this ubiquitous fungus at the point-of-care, and could help to improve clinical decision making, infection control and epidemiological surveillance.

Development and validation of LAMP primer sets for rapid identification of Aspergillus fumigatus carrying the cyp51A TR46 azole resistance gene

“…Recently Yu Shan-Ling et al 18 reported a similar rapid technique to detect azole-resistant strains due to amplification of a TR of a 34 bp (TR 34 ) and a 46 bp (TR 46 ) within the promoter region of cyp51A of A. fumigatus. However, here we used a newly designed TR 46 LAMP primer set different from those reported by Yu Shan-Ling et al 18 .…”

“…Recently Yu Shan-Ling et al 18 reported a similar rapid technique to detect azole-resistant strains due to amplification of a TR of a 34 bp (TR 34 ) and a 46 bp (TR 46 ) within the promoter region of cyp51A of A. fumigatus. However, here we used a newly designed TR 46 LAMP primer set different from those reported by Yu Shan-Ling et al 18 . Compared to experiments such as Yu Shan-Ling et al 18 , our method used adjusted genomic DNA (2 ng/μL) and can detect TR 46 strains when resistance is confirmed, and this leads to a simple identification method of A. fumigatus carrying the TR 46 in the cyp51A promoter region, in routine clinical practice.…”

“…However, here we used a newly designed TR 46 LAMP primer set different from those reported by Yu Shan-Ling et al 18 . Compared to experiments such as Yu Shan-Ling et al 18 , our method used adjusted genomic DNA (2 ng/μL) and can detect TR 46 strains when resistance is confirmed, and this leads to a simple identification method of A. fumigatus carrying the TR 46 in the cyp51A promoter region, in routine clinical practice.…”

Fungal Genomics in Respiratory Medicine: What, How and When?

“…The addition of loop primers is also able to significantly reduce the LAMP amplification time, by up to 30 min [ 177 ]. Recently, a LAMP-based assay was developed that can detect A. fumigatus containing the TR 34 and TR 46 azole resistance allele with high sensitivity, positively detecting down to 10 genomic copies/reaction in under 30 min [ 178 , 179 ]. This assay was developed so it could be performed on a “lab-on-chip” platform, a diagnostic platform which uses a silicon nitride layer to bind protons released during the incorporation of nucleic acids into DNA to track DNA amplification.…”

Application of Lab-on-Chip for Detection of Microbial Nucleic Acid in Food and Environment

“…The entire analysis process took about 4 h and 40 min, and the detection limit was as low as about 27 cells. LAMP has also been used in LOC applications for Vibrio parahaemolyticus in aquatic products, Aspergillus fumigata in clinical and environmental isolates, and Salmonella in food ( Pang et al, 2017 ; Wang et al, 2020 ; Yu L. S. et al, 2020 ).…”