Molecular Diagnosis of Invasive Aspergillosis and Detection of Azole Resistance by a Newly Commercialized PCR Kit
ABSTRACTAspergillus fumigatusis the main species responsible for aspergillosis in humans. The diagnosis of aspergillosis remains difficult, and the rapid emergence of azole resistance inA. fumigatusis worrisome. The aim of this study was to validate the new MycoGENIEA. fumigatusreal-time PCR kit and to evaluate its performance on clinical samples for the detection ofA. fumigatusand its azole resistance. This multiplex assay detects DNA from theA. fumigatusspecies complex by targeting the multicopy 28S rRNA gene and specific TR34and L98H mutations in the single-copy-numbercyp51Agene ofA. fumigatus. The specificity ofcyp51Amutation detection was assessed by testing DNA samples from 25 wild-type or mutated clinicalA. fumigatusisolates. 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 13 patients without aspergillosis. The limit of detection was <1 copy for theAspergillus28S rRNA gene and 6 copies for thecyp51Agene harboring the TR34and L98H alterations. No cross-reactivity was detected with various fungi and bacteria. All isolates harboring the TR34and L98H mutations were accurately detected by quantitative PCR (qPCR) analysis. With respiratory samples, qPCR results showed a sensitivity and specificity of 92.9% and 90.1%, respectively, while with 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 ofA. fumigatusDNA and azole resistance due to TR34and L98H mutations in clinical samples.