{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T15:15:32Z","timestamp":1790003732068,"version":"4.0.1"},"reference-count":118,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T00:00:00Z","timestamp":1753833600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Background: Polymerase chain reaction (PCR) is highly sensitive and specific for the rapid diagnosis of invasive fungal disease (IFD) but is not yet widely implemented due to concerns regarding limited standardisation between assays, the lack of commercial options and the absence of clear guidance on interpreting results. Objectives and Methods: This review provides an update on technical and clinical aspects of PCR for the diagnosis of the most pertinent fungal pathogens, including Aspergillus, Candida, Pneumocystis jirovecii, Mucorales spp., and endemic mycoses. Summary: Recent meta-analyses have demonstrated that quantitative PCR (qPCR) offers high sensitivity for diagnosing IFD, surpassing conventional microscopy, culture and most serological tests. The reported specificity of qPCR is likely underestimated due to comparison with imperfect reference standards with variable sensitivity. Although the very low limit of detection of qPCR can generate false positive results due to procedural contamination or patient colonisation (particularly in pulmonary specimens), the rates are comparable to those observed for biomarker testing. When interpreting qPCR results, it is essential to consider the pre-test probability, determined by the patient population, host factors, clinical presentation and risk factors. For patients with low to moderate pre-test probability, the use of sensitive molecular tests, often in conjunction with serological testing or biomarkers, can effectively exclude IFD when all tests return negative results, reducing the need for empirical antifungal therapy. Conversely, for patients with high pre-test probability and clinical features of IFD, qPCR testing on invasive specimens from the site of infection (such as tissue or bronchoalveolar lavage fluid) can confidently rule in the disease. The development of next-generation sequencing methods to detect fungal infection has the potential to enhance the diagnosis of IFD, but standardisation and optimisation are essential, with improved accessibility underpinning clinical utility.<\/jats:p>","DOI":"10.3390\/diagnostics15151909","type":"journal-article","created":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T10:55:37Z","timestamp":1753872937000},"page":"1909","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":22,"title":["The Molecular Diagnosis of Invasive Fungal Diseases with a Focus on PCR"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9896-8682","authenticated-orcid":false,"given":"Lottie","family":"Brown","sequence":"first","affiliation":[{"name":"Institute of Infection and Immunity, St. George\u2019s University and St. Georges University Hospitals NHS Foundation Trust, London SW17 0QT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1641-7367","authenticated-orcid":false,"given":"Mario","family":"Cruciani","sequence":"additional","affiliation":[{"name":"Fungal PCR Initiative, A Working Group of the International Society of Human and Animal Mycology, 37100 Verona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1702-0545","authenticated-orcid":false,"given":"Charles Oliver","family":"Morton","sequence":"additional","affiliation":[{"name":"School of Science, Western Sydney University, Sydney 2000, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9726-3082","authenticated-orcid":false,"given":"Alexandre","family":"Alanio","sequence":"additional","affiliation":[{"name":"Laboratoire de Parasitologie-Mycologie, AP-HP, H\u00f4pital Saint-Louis, 75010 Paris, France"},{"name":"Translational Mycology Research Group, Mycology Department, Institut Pasteur, Universit\u00e9 Paris Cit\u00e9, National Reference Center for Invasive Mycoses and Antifungals, 75015 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rosemary A.","family":"Barnes","sequence":"additional","affiliation":[{"name":"Department of Infection, University of Cardiff, Cardiff CF10 3AT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8521-335X","authenticated-orcid":false,"given":"J. Peter","family":"Donnelly","sequence":"additional","affiliation":[{"name":"The Foundation European <i>Aspergillus<\/i> PCR Initiative, de Hoefkamp 1096, 6545 MD Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5622-1916","authenticated-orcid":false,"given":"Ferry","family":"Hagen","sequence":"additional","affiliation":[{"name":"Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands & Department of Medical Microbiology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebecca","family":"Gorton","sequence":"additional","affiliation":[{"name":"Department of Infection Sciences, Health Services Laboratories [HSL] LLP, London WC1H 9AX, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9073-2886","authenticated-orcid":false,"given":"Michaela","family":"Lackner","sequence":"additional","affiliation":[{"name":"Insitute of Hygiene and Medical Microbiology, Medical University of Innsbruck, 6020 Innsbruck, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juergen","family":"Loeffler","sequence":"additional","affiliation":[{"name":"Medizinische Klinik II, Labor W\u00dc4i, Universit\u00e4tsklinikum W\u00fcrzburg, 97080 W\u00fcrzburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5939-3409","authenticated-orcid":false,"given":"Laurence","family":"Millon","sequence":"additional","affiliation":[{"name":"Parasitology-Mycology Department, University Hospital of Besanc\u0327on, Besanc\u0327on, France and UMR 6249 CNRS Chrono-Environnement, University of Franche-Comt\u00e9, 25030 Besanc\u0327on, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1071-6040","authenticated-orcid":false,"given":"Riina","family":"Rautemaa-Richardson","sequence":"additional","affiliation":[{"name":"Mycology Reference Centre Manchester and Department of Infectious Diseases, Manchester Academic Health Science Centre, Wythenshawe Hospital, Manchester University NHS Foundation Trust and Division of Evolution, Manchester M23 9LT, UK"},{"name":"Infection and Genomics, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3056-4205","authenticated-orcid":false,"given":"P. Lewis","family":"White","sequence":"additional","affiliation":[{"name":"Mycology Reference Laboratory, Public Health Wales, Microbiology Cardiff, UHW, Heath Park, Cardiff CF14 4XW, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e428","DOI":"10.1016\/S1473-3099(23)00692-8","article-title":"Global incidence and mortality of severe fungal disease","volume":"24","author":"Denning","year":"2024","journal-title":"Lancet Infect. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1127","DOI":"10.1038\/s41564-022-01172-2","article-title":"COVID-19-associated fungal infections","volume":"7","author":"Hoenigl","year":"2022","journal-title":"Nat. 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