Rapid and sensitive detection of azole-resistant Aspergillus fumigatus by tandem-repeat loop-mediated isothermal amplification
File(s)
Accepted version
Author(s)
Type
Journal Article
Abstract
Invasive human fungal infections caused by multi-azole resistant Aspergillus fumigatus are associated with increasing rates of mortality in susceptible patients. Current methods of diagnosing infections caused by multi-azole 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 multi-azole 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.
Date Issued
2019-03-01
Date Acceptance
2018-10-16
Citation
Journal of Molecular Diagnostics, 2019, 21 (2), pp.286-295
ISSN
1525-1578
Publisher
American Society for Investigative Pathology (ASIP)
Start Page
286
End Page
295
Journal / Book Title
Journal of Molecular Diagnostics
Volume
21
Issue
2
Copyright Statement
© 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. This manuscript is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International Licence http://creativecommons.org/licenses/by-nc-nd/4.0/
Sponsor
Engineering & Physical Science Research Council (E
Engineering & Physical Science Research Council (EPSRC)
Natural Environment Research Council (NERC)
Medical Research Council (MRC)
Natural Environment Research Council [2006-2012]
Wellcome Trust
Wellcome Trust
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/30529128
PII: S1525-1578(18)30208-3
Grant Number
EP/P510798/1
EP/R512655/1
NE/P001165/1
MR/R015600/1
NE/P001165/1
WTSA MMFI Clinical PhD Fellow
204566/Z/16/Z
Subjects
Science & Technology
Life Sciences & Biomedicine
Pathology
POLYMERASE-CHAIN-REACTION
REAL-TIME PCR
LINKED-IMMUNOSORBENT-ASSAY
LATERAL-FLOW DEVICE
INVASIVE ASPERGILLOSIS
GALACTOMANNAN ANTIGEN
VISUAL DETECTION
CLINICAL-USE
DIAGNOSIS
DNA
1108 Medical Microbiology
Pathology
Publication Status
Published
Coverage Spatial
United States
Date Publish Online
2018-12-04