title: SNaPAfu: a novel single nucleotide polymorphism multiplex assay for aspergillus fumigatus direct detection, identification and genotyping in clinical specimens.
abstract: OBJECTIVE
  Early diagnosis of invasive aspergillosis is essential for positive patient outcome. Likewise genotyping of fungal isolates is desirable for outbreak control in clinical setting. We designed a molecular assay that combines detection, identification, and genotyping of Aspergillus fumigatus in a single reaction.
  
  
  METHODS
  To this aim we combined 20 markers in a multiplex reaction and the results were seen following mini-sequencing readings. Pure culture extracts were firstly tested. Thereafter, Aspergillus-DNA samples obtained from clinical specimens of patients with possible, probable, or proven aspergillosis according to European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria.
  
  
  RESULTS
  A new set of designed primers allowed multilocus sequence typing (MLST) gene amplification in a single multiplex reaction. The newly proposed SNaPAfu assay had a specificity of 100%, a sensitivity of 89% and detection limit of 1 ITS copy/mL (∼0.5 fg genomic Aspergillus-DNA/mL). The marker A49_F was detected in 89% of clinical samples. The SNaPAfu assay was accurately performed on clinical specimens using only 1% of DNA extract (total volume 50 µL) from 1 mL of used bronchoalveolar lavage.
  
  
  CONCLUSIONS
  The first highly sensitive and specific, time- and cost-economic multiplex assay was implemented that allows detection, identification, and genotyping of A. fumigatus strains in a single amplification followed by mini-sequencing reaction. The new test is suitable to clinical routine and will improve patient management.
authors.affiliation: Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal ; Faculty of Sciences, University of Porto, Porto, Portugal.
authors.forename: Rita
authors.initials: R
authors.lastname: Caramalho
authors.forename: Leonor
authors.initials: L
authors.lastname: Gusmão
authors.forename: Michaela
authors.initials: M
authors.lastname: Lackner
authors.forename: António
authors.initials: A
authors.lastname: Amorim
authors.forename: Ricardo
authors.initials: R
authors.lastname: Araujo
mesh_terms: D000328:Adult; D000368:Aged; D001228:Aspergillosis / Q000175:diagnosis*; D001232:Aspergillus fumigatus / Q000235:genetics*; D005260:Female; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D058885:Multilocus Sequence Typing / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity