SNaPAfu: A Novel Single Nucleotide Polymorphism Multiplex Assay for Aspergillus fumigatus Direct Detection, Identification and Genotyping in Clinical Specimens: e75968

• Published in: PloS one 2013-10, Vol.8 (10)
• Main Authors: Caramalho, Rita, Gusmao, Leonor, Lackner, Michaela, Amorim, Antonio, Araujo, Ricardo
• Format: Article
• Language: English
• Subjects: Alveoli; Aspergillus fumigatus
• ISSN: 1932-6203
• DOI: 10.1371/journal.pone.0075968

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 mu L) 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.