{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,7]],"date-time":"2025-11-07T19:19:10Z","timestamp":1762543150284,"version":"build-2065373602"},"reference-count":23,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2020,1,10]],"date-time":"2020-01-10T00:00:00Z","timestamp":1578614400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JoF"],"abstract":"<jats:p>Diagnosis of invasive fungal infections is complex, and the lack of standardization of molecular methods is still a challenge. Several methods are available for the diagnosis of invasive aspergillosis, but their effectiveness will depend on the studied population, the patients\u2019 comorbidities, and the use of mold active prophylaxis, among others. The ability to determine the identity of the infecting Aspergillus species, and to detect mutations conferring specific resistance patterns directly from DNA extracted from the biological product, is an advantage of nucleic acid testing compared with antigen-based assays. In this study, we present laboratory cases where the diagnosis of aspergillosis was performed using a real-time multiplex PCR for the detection of Aspergillus DNA in tissue samples, showing its usefulness as one more tool in the diagnosis of aspergillosis in tissue samples. Aspergillus real-time multiplex PCR was also used to detect azole-resistance in some cases. In the majority of the PCR positive cases, cultures remained negative after 60 days. The PCR assay directed to Aspergillus gave positive signals for Aspergillus fumigatus sensu stricto. Results were confirmed by panfungal PCR, followed by sequencing, revealing 100% homology with Aspergillus fumigatus sensu stricto. Mutations conferring azole resistance were not detected.<\/jats:p>","DOI":"10.3390\/jof6010011","type":"journal-article","created":{"date-parts":[[2020,1,10]],"date-time":"2020-01-10T10:20:29Z","timestamp":1578651629000},"page":"11","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Molecular Detection of Aspergillus: Application of a Real-Time PCR Multiplex Assay in Tissue Samples"],"prefix":"10.3390","volume":"6","author":[{"given":"Raquel","family":"Sabino","sequence":"first","affiliation":[{"name":"Reference Unit for Parasitic and Fungal Infections, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisbon, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Helena","family":"Sim\u00f5es","sequence":"additional","affiliation":[{"name":"Reference Unit for Parasitic and Fungal Infections, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisbon, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Cristina","family":"Ver\u00edssimo","sequence":"additional","affiliation":[{"name":"Reference Unit for Parasitic and Fungal Infections, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisbon, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,1,10]]},"reference":[{"key":"ref_1","first-page":"195","article-title":"Opportunistic invasive fungal infections: Diagnosis & clinical management","volume":"139","author":"Badiee","year":"2014","journal-title":"Indian J. Med. Res."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Silva de Loreto, \u00c9., and Tondolo, J.S.M. (2019). Introductory Chapter: Epidemiology of Invasive Fungal Infection\u2014An Overview. Fungal Infection, IntechOpen. Available online: https:\/\/www.intechopen.com\/books\/fungal-infection\/introductory-chapter-epidemiology-of-invasive-fungal-infection-an-overview.","DOI":"10.5772\/intechopen.85955"},{"key":"ref_3","unstructured":"(2019, November 03). Fungal Infections. Available online: http:\/\/www.life-worldwide.org\/fungal-diseases\/invasive-aspergillosis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1813","DOI":"10.1086\/588660","article-title":"Revised Definitions of Invasive Fungal Disease from the European Organization for Research and Treatment of Cancer\/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC\/MSG) Consensus Group","volume":"46","author":"Thomas","year":"2008","journal-title":"Clin. Infect. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"9405","DOI":"10.1155\/2013\/459405","article-title":"Laboratory Diagnosis of Invasive Aspergillosis: From Diagnosis to Prediction of Outcome","volume":"2013","author":"Barton","year":"2013","journal-title":"Scientifica"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"S155","DOI":"10.1093\/mmy\/myy103","article-title":"How to make a fast diagnosis in invasive aspergillosis","volume":"57","year":"2019","journal-title":"Med. Mycol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Snelders, E., van der Lee, H.A., Kuijpers, J., Rijs, A.J., Varga, J., Samson, R.A., Mellado, E., Donders, A.R., Melchers, W.J., and Verweij, P.E. (2008). Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLoS Med., 5.","DOI":"10.4016\/8254.01"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"697","DOI":"10.2217\/fmb.14.27","article-title":"Exploring azole antifungal drug resistance in Aspergillus fumigatus with special reference to resistance mechanisms","volume":"9","author":"Chowdhary","year":"2014","journal-title":"Future Microbiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"515","DOI":"10.3389\/fmicb.2018.00515","article-title":"Molecular Detection of Azole-Resistant Aspergillus fumigatus in Clinical Samples","volume":"9","author":"Buil","year":"2018","journal-title":"Front. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Buil, J.B., Hagen, F., Chowdhary, A., Verweij, P.E., and Meis, J.F. (2018). Itraconazole, voriconazole, and posaconazole CLSI MIC distributions for wild-type and azole-resistant Aspergillus fumigatus isolates. J. Fungi, 4.","DOI":"10.3390\/jof4030103"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1016\/j.fgb.2015.06.001","article-title":"Genotype-phenotype complexity of the TR46\/Y121F\/T289A cyp51A azole resistance mechanism in Aspergillus fumigatus","volume":"82","author":"Snelders","year":"2015","journal-title":"Fungal Genet. Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1177\/2040620716656381","article-title":"Recent developments in the management of invasive fungal infections in patients with oncohematological diseases","volume":"7","author":"Ruhnke","year":"2016","journal-title":"Ther. Adv. Hematol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e1","DOI":"10.1016\/j.cmi.2018.01.002","article-title":"Diagnosis and management of Aspergillus diseases: Executive summary of the 2017 ESCMID-ECMM-ERS guideline","volume":"24","author":"Ullmann","year":"2018","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"750e70","DOI":"10.1093\/cid\/ciq206","article-title":"B-d-glucan assay for the diagnosis of invasive fungal infections: A meta-analysis","volume":"52","author":"Karageorgopoulos","year":"2011","journal-title":"Clin. Infect. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2655","DOI":"10.1128\/JCM.00818-16","article-title":"Molecular Diagnostic Testing for Aspergillus","volume":"54","author":"Hanson","year":"2016","journal-title":"J. Clin. Microbiol."},{"key":"ref_16","unstructured":"Reyes-Montes, M.R., Duarte-Escalante, E., Fr\u00edas-De-Le\u00f3n, M.G., Mart\u00ednez-Herrera, E.O., and Acosta-Altamirano, G. (2018). Molecular Diagnosis of Invasive Aspergillosis. Molecular Medicine, InTech Open."},{"key":"ref_17","unstructured":"Clinical and Laboratory Standards Institute (CLSI) (2012). Principles and Procedures for Detection of Fungi in Clinical Specimens-Direct Examination and Culture. Approved Guideline, CLSI Document M54-A, Clinical and Laboratory Standards Institute."},{"key":"ref_18","unstructured":"Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"4673","DOI":"10.1093\/nar\/22.22.4673","article-title":"Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice","volume":"22","author":"Thompson","year":"1994","journal-title":"Nucleic Acids Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"158","DOI":"10.3201\/eid2201.151308","article-title":"Azole resistance of Aspergillus fumigatus in immunocompromised patients with invasive Aspergillosis","volume":"22","author":"Arendrup","year":"2016","journal-title":"Emerg. Infect. Dis."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"765","DOI":"10.1139\/w11-064","article-title":"Detection of Aspergillus in lung and other tissue samples using the MycAssay Aspergillus real-time PCR kit","volume":"57","author":"Follett","year":"2011","journal-title":"Can. J. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4178","DOI":"10.1128\/JCM.02387-13","article-title":"Aspergillus PCR-based investigation of fresh tissue and effusion samples in patients with suspected invasive Aspergillosis enhances diagnostic capabilities","volume":"51","author":"Reinwald","year":"2013","journal-title":"J. Clin. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1656","DOI":"10.3389\/fmicb.2018.01656","article-title":"Aspergillus Species and Antifungals Susceptibility in Clinical Setting in the North of Portugal: Cryptic Species and Emerging Azoles Resistance in A. fumigatus","volume":"9","author":"Pinto","year":"2018","journal-title":"Front. Microbiol."}],"container-title":["Journal of Fungi"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2309-608X\/6\/1\/11\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T13:29:16Z","timestamp":1760362156000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2309-608X\/6\/1\/11"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,1,10]]},"references-count":23,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2020,3]]}},"alternative-id":["jof6010011"],"URL":"https:\/\/doi.org\/10.3390\/jof6010011","relation":{},"ISSN":["2309-608X"],"issn-type":[{"type":"electronic","value":"2309-608X"}],"subject":[],"published":{"date-parts":[[2020,1,10]]}}}