Culture independent detection of Aspergillus fumigatus Cyp51A in human lung

Aiah Khateba,b, Michael Bromleyb and Paul Bowyerb

Author address: 

aFaculaty of Applied Sciences, Medical Laboratory Technology, University of Taibah, Medina, Kingdom of Saudi Arabia bFungal Infection Group, Centre for Respiratory Medicine and Allergy, Faculty of Biology, Medicine and Health,Institute of Inflammation and Repair, Manchester Academic Health Science Centre, The University of Manchester and University Hospital of South Manchester

Abstract: 

Background: Aspergillus fumigatus is the main causative agent of aspergillosis. Traditional culture methods recover a small fraction of all the lung resident species and therefore resistant isolates are usually missed. Long-term triazole oral therapy, particularly itraconazole and voriconazole has been used in treatment of chronic pulmonary Aspergillosis or allergic aspergillosis. Azole resistance is associated with poorer disease outcome in allergic and chronic disease and has increased in recent years. This study aims to detect resistant mutations in cyp51A in the context of uncultured Aspergillus strains present in clinical samples.

Methods: DNA was extracted directly from 38 BAL samples (8 healthy controls, 9 ABPA, 8 SAFS, 7 SA and 3 MA). The cyp51A gene was directly amplified from extracted DNA and amplicons sequenced using illumina Miseq. Sequence analysis was done using the following software pipline; trimmomatic 0.36>bowtie2>samtools1.2>bcftools htslib 1.3.1.>VarScan.

Results: 61 SNPS were identified, 29 resulted in amino acid change. Three SNPs matched cyp51A mutations that have previously been shown to confer azole resistance; M220I/M220T and F46L. 9 further mutations occurred only in azole treated patients; N218T, F219Y, N218Y, M220T, M220I, I217S, A62C, I135T, T112I and 17 were common to sequences obtained from patients with no azole treatment history.

Conclusions: This study has identified new cyp51A mutations in the healthy, chronically colonized patients. It also broadened our understanding of cyp51A resistance mechanisms in the context of uncultured Aspergillus strains present in clinical samples.

2017

abstract No: 

37

Full conference title: 

The Fourteenth International Aspergillus Meeting, Asilomar Conference Center, Pacific Grove, CA, USA
    • Asperfest 14 (2017)