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

• Tipo: Artigo em Revista Científica Internacional
• Data: 2013

Autores

Rita Caramalho (Autor) Outra; Leonor Gusmao (Autor) Outra; Michaela Lackner (Autor) Outra; Antonio Amorim (Autor) FCUP; Ricardo Araujo (Autor) Outra

Revista

Título: PLoS ONE

Vol. 8 Nº 1

Página Final: e75968

ISSN: 1932-6203

Editora: Public Library of Science

Indexação

ISI Web of Knowledge - 14 Citações

Scopus - 11 Citações

Outras Informações

ID Authenticus: P-008-F8B

DOI: 10.1371/journal.pone.0075968

Abstract (EN): 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 (similar to 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.

Idioma: Inglês

Tipo (Avaliação Docente): Científica

Nº de páginas: 9