5 years ago

Molecular diagnosis of invasive aspergillosis and detection of azole resistance by a newly commercialized PCR kit.

Quesne G, Gabriel F, Accoceberry I, Godichaud S, Lagardere G, Verweij PE, Audebert L, Gaboyard M, Dannaoui E, Bougnoux ME
Aspergillus fumigatus is the main species responsible for aspergillosis in humans. The diagnosis of aspergillosis remains difficult, and the rapid emergence of azole resistance in A. fumigatus, is worrisome. The aim of this study was to validate the new MycoGENIE® A. fumigatus real-time PCR kit and to evaluate its performance in clinical samples for the detection of A. fumigatus and its azole resistance. This multiplex assay detects DNA from the A. fumigatus species complex by targeting the 28S-rRNA multicopy gene and specific TR34 and L98H mutations in the cyp51A single copy gene of A. fumigatus The specificity of cyp51A mutation detection was assessed by testing DNA samples from 25 wild-type or mutated clinical A. fumigatus isolates. Clinical validation was performed on 88 respiratory samples obtained from 62 patients, and on 69 serum samples obtained from 16 patients with proven or probable aspergillosis and from 13 patients without aspergillosis. The limit of detection was below one copy for the Aspergillus 28S-rRNA gene and six copies for the cyp51A gene harboring the TR34/L98H alterations. No cross-reactivity was detected with various fungi and bacteria. All isolates harboring the TR34/L98H mutations were accurately detected by qPCR analysis. In respiratory samples, qPCR results showed a sensitivity and specificity of 92.9% and 90.1%, respectively, while in serum samples, the sensitivity and specificity were 100% and 84.6%, respectively. Our study demonstrated that this new real-time PCR kit enables sensitive and rapid detection of A. fumigatus DNA and azole resistance due to TR34/L98H mutations in clinical samples.

Publisher URL: https://www.ncbi.nlm.nih.gov/pubmed/28814586

DOI: PubMed:28814586

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