title: High-frequency triazole resistance found In nonculturable Aspergillus fumigatus from lungs of patients with chronic fungal disease.
abstract: BACKGROUND
  Oral triazole therapy is well established for the treatment of invasive (IPA), allergic (ABPA), and chronic pulmonary (CPA) aspergillosis, and is often long-term. Triazole resistance rates are rising internationally. Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequency of triazole resistance.
  
  
  METHODS
  Using an ultrasensitive real-time polymerase chain reaction (PCR) assay for Aspergillus spp., we assessed respiratory fungal load in bronchoalveolar lavage (BAL) and sputum specimens. In a subset of PCR-positive, culture negative samples, we further amplified the CYP51A gene to detect key single-nucleotide polymorphisms (SNPs) associated with triazole resistance.
  
  
  RESULTS
  Aspergillus DNA was detected in BAL from normal volunteers (4/11, 36.4%) and patients with culture or microscopy confirmed IPA (21/22, 95%). Aspergillus DNA was detected in sputum in 15 of 19 (78.9%) and 30 of 42 (71.4%) patients with ABPA and CPA, compared with 0% and 16.7% by culture, respectively. In culture-negative, PCR-positive samples, we detected triazole-resistance mutations (L98H with tandem repeat [TR] and M220) within the drug target CYP51A in 55.1% of samples. Six of 8 (75%) of those with ABPA and 12 of 24 (50%) with CPA had resistance markers present, some without prior triazole treatment, and in most despite adequate plasma drug concentrations around the time of sampling.
  
  
  CONCLUSIONS
  The very low organism burdens of fungi causing infection have previously prevented direct culture and detection of antifungal resistance in clinical samples. These findings have major implications for the sustainability of triazoles for human antifungal therapy.
authors.affiliation: National Aspergillosis Centre, University of Manchester, Manchester, UK. ddenning@manchester.ac.uk
authors.forename: David W
authors.initials: DW
authors.lastname: Denning
authors.forename: Steven
authors.initials: S
authors.lastname: Park
authors.forename: Cornelia
authors.initials: C
authors.lastname: Lass-Florl
authors.forename: Marcin G
authors.initials: MG
authors.lastname: Fraczek
authors.forename: Marie
authors.initials: M
authors.lastname: Kirwan
authors.forename: Robin
authors.initials: R
authors.lastname: Gore
authors.forename: Jaclyn
authors.initials: J
authors.lastname: Smith
authors.forename: Ahmed
authors.initials: A
authors.lastname: Bueid
authors.forename: Caroline B
authors.initials: CB
authors.lastname: Moore
authors.forename: Paul
authors.initials: P
authors.lastname: Bowyer
authors.forename: David S
authors.initials: DS
authors.lastname: Perlin
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001229:Aspergillosis, Allergic Bronchopulmonary / Q000188:drug therapy / Q000453:epidemiology / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D002908:Chronic Disease; D003470:Culture Media; D004271:DNA, Fungal / Q000032:analysis; D025141:Drug Resistance, Fungal / Q000235:genetics; D006801:Humans; D055744:Invasive Pulmonary Aspergillosis / Q000188:drug therapy / Q000453:epidemiology / Q000382:microbiology; D008168:Lung / Q000382:microbiology*; D009154:Mutation; D016133:Polymerase Chain Reaction; D055732:Pulmonary Aspergillosis / Q000188:drug therapy / Q000453:epidemiology* / Q000382:microbiology; D013183:Sputum / Q000382:microbiology; D014230:Triazoles / Q000494:pharmacology* / Q000627:therapeutic use
chemical_list: D000935:Antifungal Agents; D003470:Culture Media; D004271:DNA, Fungal; D014230:Triazoles