{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T03:37:38Z","timestamp":1789529858653,"version":"build-2803163510"},"reference-count":31,"publisher":"American Society for Microbiology","issue":"7","license":[{"start":{"date-parts":[[2012,7,1]],"date-time":"2012-07-01T00:00:00Z","timestamp":1341100800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Antimicrob Agents Chemother"],"published-print":{"date-parts":[[2012,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title><jats:p>The increasing incidence of azole resistance in<jats:named-content xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" content-type=\"genus-species\" xlink:type=\"simple\">Aspergillus fumigatus<\/jats:named-content>causing invasive aspergillosis (IA) in immunocompromised\/hematological patients emphasizes the need to improve the detection of resistance-mediating<jats:italic>cyp51A<\/jats:italic>gene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on a positive culture yield in this group of patients. We generated primers from the unique sequence of the<jats:italic>Aspergillus fumigatus cyp51A<\/jats:italic>gene to establish PCR assays with consecutive DNA sequence analysis to detect and identify the<jats:italic>A. fumigatus cyp51A<\/jats:italic>tandem repeat (TR) mutation in the promoter region and the L98H and M220 alterations directly in clinical samples. After testing of the sensitivity and specificity of the assays using serially diluted<jats:named-content xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" content-type=\"genus-species\" xlink:type=\"simple\">A. fumigatus<\/jats:named-content>and human DNA,<jats:italic>A. fumigatus cyp51A<\/jats:italic>gene fragments of about 150 bp potentially carrying the mutations were amplified directly from primary clinical samples and subsequently DNA sequenced. The determined sensitivities of the PCR assays were 600 fg, 6 pg, and 4 pg of<jats:named-content xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" content-type=\"genus-species\" xlink:type=\"simple\">A. fumigatus<\/jats:named-content>DNA for the TR, L98H, and M220 mutations, respectively. There was no cross-reactivity with human genomic DNA detectable. Sequencing of the PCR amplicons for<jats:named-content xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" content-type=\"genus-species\" xlink:type=\"simple\">A. fumigatus<\/jats:named-content>wild-type DNA confirmed the<jats:italic>cyp51A<\/jats:italic>wild-type sequence, and PCR products from one azole-resistant<jats:named-content xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" content-type=\"genus-species\" xlink:type=\"simple\">A. fumigatus<\/jats:named-content>isolate showed the L98H and TR mutations. The second azole-resistant isolate revealed an M220T alteration. We consider our assay to be of high epidemiological and clinical relevance to detect azole resistance and to optimize antifungal therapy in patients with IA.<\/jats:p>","DOI":"10.1128\/aac.05902-11","type":"journal-article","created":{"date-parts":[[2012,4,24]],"date-time":"2012-04-24T05:01:18Z","timestamp":1335243678000},"page":"3905-3910","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":58,"title":["Development of Novel PCR Assays To Detect Azole Resistance-Mediating Mutations of the<i>Aspergillus fumigatus cyp51A<\/i>Gene in Primary Clinical Samples from Neutropenic Patients"],"prefix":"10.1128","volume":"56","author":[{"given":"Birgit","family":"Spiess","sequence":"first","affiliation":[{"name":"3. 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