title: PCR-based detection of Aspergillus fumigatus Cyp51A mutations on bronchoalveolar lavage: a multicentre validation of the AsperGenius assay® in 201 patients with haematological disease suspected for invasive aspergillosis.
abstract: In patients with invasive aspergillosis (IA), fungal cultures are mostly negative. Consequently, azole resistance often remains undetected. The AsperGenius®multiplex real-time PCR assay identifies clinically relevant Aspergillus species and four resistance-associated mutations (RAMs; TR34/L98H/T289A/Y121F) in the Cyp51A gene. This multicentre study evaluated the diagnostic performance of this assay on bronchoalveolar lavage (BAL) fluid and correlated the presence of RAMs with azole treatment failure and mortality.
  
  
  
  Stored BAL samples from patients with haematological diseases with suspected IA were used. BAL samples that were galactomannan/culture positive were considered positive controls for the presence of Aspergillus. Azole treatment failure and 6 week mortality were compared in patients with and without RAMs that had received ≥5 days of voriconazole monotherapy.
  
  
  
  Two hundred and one patients each contributed one BAL sample, of which 88 were positive controls and 113 were negative controls. The optimal cycle threshold cut-off value for the Aspergillus species PCR was <38. With this cut-off, the PCR was positive in 74/88 positive controls. The sensitivity, specificity, positive predictive value and negative predictive value were 84%, 80%, 76% and 87%, respectively. 32/74 BAL samples were culture negative. Azole treatment failure was observed in 6/8 patients with a RAM compared with 12/45 patients without RAMs (P = 0.01). Six week mortality was 2.7 times higher in patients with RAMs (50.0% versus 18.6%; P = 0.07).
  
  
  
  The AsperGenius®assay had a good diagnostic performance on BAL and differentiated WT from Aspergillus fumigatus with RAMs, including in culture-negative BAL samples. Most importantly, detection of RAMs was associated with azole treatment failure.
authors.affiliation: Department of Internal Medicine, Section of Infectious Diseases, Erasmus University Medical Center, Rotterdam, The Netherlands g.chong@erasmusmc.nl.
authors.forename: G M
authors.initials: GM
authors.lastname: Chong
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: M T
authors.initials: MT
authors.lastname: van der Beek
authors.affiliation: Department of Haematology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: P A
authors.initials: PA
authors.lastname: von dem Borne
authors.affiliation: Department of Microbiology, Ghent University Hospital, Ghent, Belgium.
authors.forename: J
authors.initials: J
authors.lastname: Boelens
authors.affiliation: Department of Haematology, Ghent University Hospital, Ghent, Belgium.
authors.forename: E
authors.initials: E
authors.lastname: Steel
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: G A
authors.initials: GA
authors.lastname: Kampinga
authors.affiliation: Department of Haematology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: L F R
authors.initials: LF
authors.lastname: Span
authors.affiliation: Department of Microbiology and Immunology, KU Leuven-University of Leuven; University Hospitals Leuven, Leuven, Belgium.
authors.forename: K
authors.initials: K
authors.lastname: Lagrou
authors.affiliation: Department of Microbiology and Immunology, KU Leuven-University of Leuven; Department of Haematology, University Hospitals Leuven, Leuven, Belgium.
authors.forename: J A
authors.initials: JA
authors.lastname: Maertens
authors.affiliation: PathoNostics B.V., Maastricht, The Netherlands.
authors.forename: G J H
authors.initials: GJ
authors.lastname: Dingemans
authors.affiliation: PathoNostics B.V., Maastricht, The Netherlands.
authors.forename: G R
authors.initials: GR
authors.lastname: Gaajetaan
authors.affiliation: PathoNostics B.V., Maastricht, The Netherlands.
authors.forename: D W E
authors.initials: DW
authors.lastname: van Tegelen
authors.affiliation: Department of Haematology, Erasmus University Medical Center, Rotterdam, The Netherlands.
authors.forename: J J
authors.initials: JJ
authors.lastname: Cornelissen
authors.affiliation: Department of Medical Microbiology, Erasmus University Medical Center, Rotterdam, The Netherlands.
authors.forename: A G
authors.initials: AG
authors.lastname: Vonk
authors.affiliation: Department of Internal Medicine, Section of Infectious Diseases, Erasmus University Medical Center, Rotterdam, The Netherlands.
authors.forename: B J A
authors.initials: BJ
authors.lastname: Rijnders
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000935:Antifungal Agents / Q000494:pharmacology / Q000627:therapeutic use; D001232:Aspergillus fumigatus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D001393:Azoles / Q000494:pharmacology / Q000627:therapeutic use; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D025141:Drug Resistance, Fungal; D005260:Female; D005656:Fungal Proteins / Q000235:genetics*; D060005:Genotyping Techniques / Q000379:methods; D006402:Hematologic Diseases / Q000150:complications; D006801:Humans; D055744:Invasive Pulmonary Aspergillosis / Q000175:diagnosis* / Q000382:microbiology; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies; D016019:Survival Analysis; D017211:Treatment Failure; D055815:Young Adult
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D005656:Fungal Proteins; D003577:Cytochrome P-450 Enzyme System; C465526:cytochrome P-450 CYP51A, Aspergillus