title: Validation of a new Aspergillus real-time PCR assay for direct detection of Aspergillus and azole resistance of Aspergillus fumigatus on bronchoalveolar lavage fluid.
abstract: Azole resistance in Aspergillus fumigatus is increasingly reported. Here, we describe the validation of the AsperGenius, a new multiplex real-time PCR assay consisting of two multiplex real-time PCRs, one that identifies the clinically relevant Aspergillus species, and one that detects the TR34, L98H, T289A, and Y121F mutations in CYP51A and differentiates susceptible from resistant A. fumigatus strains. The diagnostic performance of the AsperGenius assay was tested on 37 bronchoalveolar lavage (BAL) fluid samples from hematology patients and 40 BAL fluid samples from intensive care unit (ICU) patients using a BAL fluid galactomannan level of ≥1.0 or positive culture as the gold standard for detecting the presence of Aspergillus. In the hematology and ICU groups combined, there were 22 BAL fluid samples from patients with invasive aspergillosis (IA) (2 proven, 9 probable, and 11 nonclassifiable). Nineteen of the 22 BAL fluid samples were positive, according to the gold standard. The optimal cycle threshold value for the presence of Aspergillus was <36. Sixteen of the 19 BAL fluid samples had a positive PCR (2 Aspergillus species and 14 A. fumigatus samples). This resulted in a sensitivity, specificity, and positive and negative predictive values of 88.9%, 89.3%, 72.7%, and 96.2%, respectively, for the hematology group and 80.0%, 93.3%, 80.0%, and 93.3%, respectively, in the ICU group. The CYP51A real-time PCR confirmed 12 wild-type and 2 resistant strains (1 TR34-L98H and 1 TR46-Y121F-T289A mutant). Voriconazole therapy failed for both patients. The AsperGenius multiplex real-time PCR assay allows for sensitive and fast detection of Aspergillus species directly from BAL fluid samples. More importantly, this assay detects and differentiates wild-type from resistant strains, even if BAL fluid cultures remain negative.
authors.affiliation: Department of Internal Medicine, Infectious Diseases, Erasmus Medical Center, Rotterdam, the Netherlands g.chong@erasmusmc.nl.
authors.forename: Ga-Lai M
authors.initials: GL
authors.lastname: Chong
authors.affiliation: Medical Microbiology and Infectious Diseases, Erasmus Medical Center, Rotterdam, the Netherlands.
authors.forename: Wendy W J
authors.initials: WW
authors.lastname: van de Sande
authors.affiliation: PathoNostics B.V., Maastricht, the Netherlands.
authors.forename: Gijs J H
authors.initials: GJ
authors.lastname: Dingemans
authors.affiliation: PathoNostics B.V., Maastricht, the Netherlands.
authors.forename: Giel R
authors.initials: GR
authors.lastname: Gaajetaan
authors.affiliation: Medical Microbiology and Infectious Diseases, Erasmus Medical Center, Rotterdam, the Netherlands.
authors.forename: Alieke G
authors.initials: AG
authors.lastname: Vonk
authors.affiliation: Department of Clinical Microbiology, University Hospital of Liège (CHU), Liège, Belgium.
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Hayette
authors.affiliation: PathoNostics B.V., Maastricht, the Netherlands.
authors.forename: Dennis W E
authors.initials: DW
authors.lastname: van Tegelen
authors.affiliation: PathoNostics B.V., Maastricht, the Netherlands.
authors.forename: Guus F M
authors.initials: GF
authors.lastname: Simons
authors.affiliation: Department of Internal Medicine, Infectious Diseases, Erasmus Medical Center, Rotterdam, the Netherlands.
authors.forename: Bart J A
authors.initials: BJ
authors.lastname: Rijnders
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D001230:Aspergillus / Q000187:drug effects / Q000302:isolation & purification*; D001393:Azoles / Q000494:pharmacology*; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D005260:Female; D005656:Fungal Proteins / Q000235:genetics; D006801:Humans; D055744:Invasive Pulmonary Aspergillosis / Q000175:diagnosis / Q000382:microbiology; D008297:Male; D008828:Microbiological Techniques / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D050505:Mutant Proteins / Q000235:genetics; D020125:Mutation, Missense; D011237:Predictive Value of Tests; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies; D012680:Sensitivity and Specificity
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D005656:Fungal Proteins; D050505:Mutant Proteins; D003577:Cytochrome P-450 Enzyme System; C465526:cytochrome P-450 CYP51A, Aspergillus