Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens

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Background: Infections caused by triazole drug-resistant Aspergillus fumigatus are an increasing problem. The sensitivity of standard culture is poor, abrogating susceptibility testing. Early detection of resistance can improve patient outcomes, yet tools for this purpose are limited. 

Objectives: To develop and validate a pyrosequencing technique to detect resistance-conferring cyp51A polymorphisms from clinical respiratory specimens and A. fumigatus isolates. 

Methods: Method validation was performed by Sanger sequencing and pyrosequencing of 50 A. fumigatus isolates with a spectrum of triazole susceptibility patterns. Then, 326 Aspergillus quantitative PCR (qPCR)-positive respiratory samples collected over a 27 month period (January 2017-March 2019) from 160 patients at the UK National Aspergillosis Centre were assessed by cyp51A pyrosequencing. The Sanger sequencing and pyrosequencing results were compared with those from high-volume culture and standard susceptibility testing. 

Results: The cyp51A genotypes of the 50 isolates analysed by pyrosequencing and Sanger sequencing matched. Of the 326 Aspergillus qPCR-positive respiratory specimens, 71.2% were reported with no A. fumigatus growth. Of these, 56.9% (132/232) demonstrated a WT cyp51A genotype and 31.5% (73/232) a resistant genotype by pyrosequencing. Pyrosequencing identified the environmental TR34/L98H mutation in 18.7% (61/326) of the samples in contrast to 6.4% (21/326) pan-azole resistance detected by culture. Importantly, pyrosequencing detected resistance earlier than culture in 23.3% of specimens. 

Conclusions: The pyrosequencing assay described could detect a wide range of cyp51A polymorphisms associated with triazole resistance, including those not identified by commercial assays. This method allowed prompt recognition of resistance and the selection of appropriate antifungal treatment when culture was negative.

Original languageEnglish
Pages (from-to)3501-3509
Number of pages9
JournalJournal of Antimicrobial Chemotherapy
Issue number12
Early online date30 Aug 2020
Publication statusPublished - 1 Dec 2020


  • Antifungal Agents/pharmacology
  • Aspergillus fumigatus/genetics
  • Azoles
  • Cytochrome P-450 Enzyme System/genetics
  • Drug Resistance, Fungal
  • Fungal Proteins/genetics
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Microbial Sensitivity Tests
  • Triazoles/pharmacology


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