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Here we aimed to study whether phenotypic variants of ARAF that co-exist in clinical specimens were genetically distinct. A panel of six phenotypic variants of ARAF cultured from two sputum samples collected from two patients with chronic aspergillosis were included. Preliminary identification of all isolates was obtained using MALDI\u2013ToF mass spectrometry and confirmed by AsperGenius<jats:sup>\u00ae<\/jats:sup>real-time PCR assay. Antifungal susceptibility testing was determined using EUCAST E.Def 9.3 microbroth dilution. Genomic DNA libraries were constructed with the Illumina TruSeq Nano kit. Prepared whole-genome libraries were sequenced on an Illumina HiSeq 2500. Whole genome data were converted into presence\/absence of a SNP with respect to the Af293 reference genome. Colonies of ARAF that co-existed in one respiratory sample demonstrated marked phenotypic diversity. Two<jats:italic>cyp51A<\/jats:italic>polymorphisms were found among azole-resistant isolates: TR<jats:sub>34<\/jats:sub>\/L98H\/T289A\/I364V\/G448S was consistently present in four variants with a pan-azole resistant phenotype and TR<jats:sub>34<\/jats:sub>\/L98H was detected in two variants (itraconazole MIC\u2009&gt;\u200916\u00a0mg\/L). WGS typing showed that despite marked phenotypic variation, each sample contained a population of highly genetically related azole-resistant<jats:italic>A. fumigatus<\/jats:italic>variants. Our SNP analysis suggest that mechanisms additional to genetic-based variation are responsible for phenotypic diversity. Our data demonstrate that the phenotypic variants of ARAF that co-exist in clinical specimens are highly clonal and strongly suggest their origination from a single common ancestor.<\/jats:p>","DOI":"10.1007\/s11046-022-00665-2","type":"journal-article","created":{"date-parts":[[2022,9,13]],"date-time":"2022-09-13T11:02:34Z","timestamp":1663066954000},"page":"497-508","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Phenotypic Variants of Azole-Resistant Aspergillus Fumigatus that Co-exist in Human Respiratory Samples are Genetically Highly Related"],"prefix":"10.1007","volume":"187","author":[{"given":"Alireza","family":"Abdolrasouli","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1338-7860","authenticated-orcid":false,"given":"Johanna L.","family":"Rhodes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,9,13]]},"reference":[{"issue":"16","key":"665_CR1","doi-asserted-by":"publisher","first-page":"1496","DOI":"10.1056\/NEJMra2027424","volume":"385","author":"GR Thompson","year":"2021","unstructured":"Thompson GR, Young J-AH. 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JR is funded by a Wellcome Trust Institutional Strategic Support Fund Springboard Fellowship.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Conflict of interest"}},{"value":"Ethical approval for this study was not required.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethical Approval"}}]}}