title: Single-tube PCR coupled with mini-sequencing assay for the detection of cyp51A and cyp51B polymorphisms in Aspergillus fumigatus.
abstract: OBJECTIVE
  Triazole resistance in Aspergillus fumigatus is associated with mutations in cyp51 genes, therefore, a single-tube multiplex PCR was proposed for rapid detection of such mutations.
  
  
  METHODS
  Relevant markers (n = 21) located in cyp51A and cyp51B were amplified in a multiplex reaction and subsequently analyzed by mini-sequencing.
  
  
  RESULTS
  A set of nonresistant A. fumigatus were tested. The markers F46, G89, M172, D255, L358, E427 and C454 located in cyp51A, as well as P394 and S35 from cyp51B, were found to be modified.
  
  
  CONCLUSIONS
  A. fumigatus triazole resistance in Portugal is rare, nevertheless, some isolates showed alterations in the cyp51 genes. Multicenter studies with more isolates should better evaluate and validate the potential use of this method in clinical laboratories. The new methodology allows the addition of extra markers if described as relevant for A. fumigatus susceptibility to triazoles.
authors.affiliation: IPATIMUP, Institute of Molecular Pathology & Immunology of the University of Porto, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal.|Instituto de Investigação e Inovação em Saúde, Universidade do Porto; Rua Júlio Amaral de Carvalho 45, 4200-135 Porto, Portugal.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Araujo
authors.affiliation: IPATIMUP, Institute of Molecular Pathology & Immunology of the University of Porto, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal.|Department of Medical Microbiology, Istanbul Faculty of Medicine, Istanbul University, Millet Street 34093, Istanbul, Turkey.
authors.forename: Ozge
authors.initials: O
authors.lastname: Gungor
authors.affiliation: IPATIMUP, Institute of Molecular Pathology & Immunology of the University of Porto, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal.|Instituto de Investigação e Inovação em Saúde, Universidade do Porto; Rua Júlio Amaral de Carvalho 45, 4200-135 Porto, Portugal.|Faculty of Sciences, University of Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal.
authors.forename: António
authors.initials: A
authors.lastname: Amorim
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D001228:Aspergillosis / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D025141:Drug Resistance, Fungal; D005656:Fungal Proteins / Q000235:genetics*; D060005:Genotyping Techniques / Q000379:methods; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D011174:Portugal; D017422:Sequence Analysis, DNA / Q000379:methods*; D014230:Triazoles / Q000494:pharmacology
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; D014230:Triazoles; D003577:Cytochrome P-450 Enzyme System; C465526:cytochrome P-450 CYP51A, Aspergillus; C465527:cytochrome P-450 CYP51B, Aspergillus
keywords: antifungal susceptibility; fungal infection; itraconazole; mold; posaconazole; triazole resistance; voriconazole