{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,17]],"date-time":"2026-10-17T20:12:02Z","timestamp":1792267922700,"version":"4.4.0"},"reference-count":31,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2022,4,29]],"date-time":"2022-04-29T00:00:00Z","timestamp":1651190400000},"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>Cryptococcus neoformans and Cryptococcus gattii are the principle causative agents of cryptococcosis. Differences in epidemiological and clinical features, and also treatment, mean it is important for diagnostic laboratories to distinguish between the two species. Molecular methods are potentially more rapid than culture and cryptococcal antigen (CRAG) detection; however, commercial PCR-based assays that target Cryptococcus do not distinguish between species. Here, we developed a real-time PCR assay targeting the multicopy mitochondrial cytochrome b (cyt b) gene to detect C. neoformans and C. gattii in clinical specimens. Assay performance was compared with culture, histopathology, CRAG and panfungal PCR\/DNA sequencing. The cyt b-directed assay accurately detected and identified all eight C. neoformans\/gattii genotypes. High-resolution melt curve analysis unambiguously discriminated between the two species. Overall, assay sensitivity (96.4%) compared favorably with panfungal PCR (76.9%) and culture (14.5%); assay specificity was 100%. Of 25 fresh frozen paraffin embedded (FFPE) specimens, assay sensitivity was 96% (76% for panfungal PCR; 68% for histopathology). The Cryptococcus-specific PCR is a rapid (~4 h) sensitive method to diagnose (or exclude) cryptococcosis and differentiate between the two major species. It is suitable for use on diverse clinical specimens and may be the preferred molecular method for FFPE specimens where clinical suspicion of cryptococcosis is high.<\/jats:p>","DOI":"10.3390\/jof8050462","type":"journal-article","created":{"date-parts":[[2022,4,30]],"date-time":"2022-04-30T05:44:07Z","timestamp":1651297447000},"page":"462","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Development of a Real-Time PCR Assay to Identify and Distinguish between Cryptococcus neoformans and Cryptococcus gattii Species Complexes"],"prefix":"10.3390","volume":"8","author":[{"given":"Enoch","family":"Tay","sequence":"first","affiliation":[{"name":"Research Education Network, Western Sydney Local Health District, Westmead Hospital, Westmead, NSW 2145, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sharon C-A.","family":"Chen","sequence":"additional","affiliation":[{"name":"Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, NSW Health Pathology, Westmead Hospital, Westmead, NSW 2145, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wendy","family":"Green","sequence":"additional","affiliation":[{"name":"Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, NSW Health Pathology, Westmead Hospital, Westmead, NSW 2145, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ronald","family":"Lopez","sequence":"additional","affiliation":[{"name":"Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, NSW Health Pathology, Westmead Hospital, Westmead, NSW 2145, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0301-7110","authenticated-orcid":false,"given":"Catriona L.","family":"Halliday","sequence":"additional","affiliation":[{"name":"Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, NSW Health Pathology, Westmead Hospital, Westmead, NSW 2145, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,4,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1133","DOI":"10.1093\/cid\/ciab268","article-title":"MSG07: An International Cohort Study Comparing Epidemiology and Outcomes of Patients With Cryptococcus neoformans or Cryptococcus gattii Infections","volume":"73","author":"Baddley","year":"2021","journal-title":"Clin. Infect. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.fgb.2015.02.009","article-title":"Recognition of seven species in the Cryptococcus gattii\/Cryptococcus neoformans species complex","volume":"78","author":"Hagen","year":"2015","journal-title":"Fungal Genet. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1367","DOI":"10.1093\/cid\/ciz1008","article-title":"Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium","volume":"71","author":"Donnelly","year":"2020","journal-title":"Clin. Infect. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1111\/imj.15590","article-title":"Consensus guidelines for the diagnosis and management of cryptococcosis and rare yeast infections in the haematology\/oncology setting, 2021","volume":"51","author":"Chang","year":"2021","journal-title":"Intern. Med. J."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Beardsley, J., Sorrell, T.C., and Chen, S.C. (2019). Central Nervous System Cryptococcal Infections in Non-HIV Infected Patients. J. Fungi, 5.","DOI":"10.3390\/jof5030071"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1016\/j.cca.2021.11.017","article-title":"Comparison of laboratory diagnosis, clinical manifestation, and management of pulmonary cryptococcosis: Report of the clinical scenario and literature review","volume":"524","author":"Hsiao","year":"2022","journal-title":"Clin. Chim. Acta"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Trilles, L., Wang, B., Firacative, C., Laz\u00e9ra, M.D.S., Wanke, B., and Meyer, W. (2014). Identification of the Major Molecular Types of Cryptococcus neoformans and C. gattii by Hyperbranched Rolling Circle Amplification. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0094648"},{"key":"ref_8","first-page":"89","article-title":"Accurate and sensitive real-time PCR assays using intergenic spacer 1 region to differentiate Cryptococcus gattii sensu lato and Cryptococcus neoformans sensu lato","volume":"54","author":"Tavares","year":"2016","journal-title":"Med. Mycol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2903","DOI":"10.3389\/fmicb.2019.02903","article-title":"A New Age in Molecular Diagnostics for Invasive Fungal Disease: Are We Ready?","volume":"10","author":"Kidd","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3021","DOI":"10.1128\/JCM.00689-08","article-title":"Multiplex tandem PCR: A novel platform for rapid detection and identification of fungal pathogens from blood culture specimens","volume":"46","author":"Lau","year":"2008","journal-title":"J. Clin. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1016\/j.cmi.2019.11.016","article-title":"Diagnostic test accuracy of the BioFire\u00ae FilmArray\u00ae meningitis\/encephalitis panel: A systematic review and meta-analysis","volume":"26","author":"Tansarli","year":"2020","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1375","DOI":"10.1016\/j.cmi.2020.01.007","article-title":"Evaluation of the Biofire FilmArray meningitis\/encephalitis assay for the detection of Cryptococcus neoformans\/gattii","volume":"26","author":"Van","year":"2020","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4058","DOI":"10.1128\/JCM.01411-08","article-title":"Colony multiplex-tandem PCR for rapid, accurate identification of fungal cultures","volume":"46","author":"Lau","year":"2008","journal-title":"J. Clin. Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"408","DOI":"10.1093\/mmy\/myz064","article-title":"False negative diagnostic errors with polymerase chain reaction for the detection of cryptococcal meningoencephalitis","volume":"58","author":"Lewis","year":"2020","journal-title":"Med. Mycol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"791","DOI":"10.1016\/j.cmi.2018.02.024","article-title":"Culture-confirmed cryptococcal meningitis not detected by Cryptococcus PCR on the Biofire meningitis\/encephalitis panel((R))","volume":"24","author":"Chew","year":"2018","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"635","DOI":"10.1016\/j.pathol.2020.09.029","article-title":"Comparison of a commercial real-time PCR panel to routine laboratory methods for the diagnosis of meningitis-encephalitis","volume":"53","author":"Chong","year":"2021","journal-title":"Pathology"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4503","DOI":"10.1128\/JCM.38.12.4503-4510.2000","article-title":"Identification and phylogenetic relationship of the most common pathogenic Candida species inferred from mitochondrial cytochrome b gene sequences","volume":"38","author":"Yokoyama","year":"2000","journal-title":"J. Clin. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"95","DOI":"10.3314\/jjmm.42.95","article-title":"Identification of Candida dubliniensis based on the specific amplification of mitochondrial cytochrome b gene","volume":"42","author":"Biswas","year":"2001","journal-title":"Nihon Ishinkin Gakkai Zasshi"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"5572","DOI":"10.1128\/JCM.41.12.5572-5576.2003","article-title":"Molecular analysis of Cryptococcus neoformans mitochondrial cytochrome b gene sequences","volume":"41","author":"Biswas","year":"2003","journal-title":"J. Clin. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"380","DOI":"10.1128\/JCM.01862-06","article-title":"Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens","volume":"45","author":"Lau","year":"2007","journal-title":"J. Clin. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"584","DOI":"10.1016\/j.pathol.2020.03.013","article-title":"Knowledge at what cost? An audit of the utility of panfungal PCR performed on bronchoalveolar lavage fluid specimens at a tertiary mycology laboratory","volume":"52","author":"Garnham","year":"2020","journal-title":"Pathology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1038\/msb.2011.75","article-title":"Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega","volume":"7","author":"Sievers","year":"2011","journal-title":"Mol. Syst. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1007\/s15010-002-3095-9","article-title":"Marginal and subgingival plaque\u2014A natural habitat of Tropheryma whipplei?","volume":"31","author":"Zinkernagel","year":"2003","journal-title":"Infection"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Firacative, C., Trilles, L., and Meyer, W. (2012). MALDI-TOF MS enables the rapid identification of the major molecular types within the Cryptococcus neoformans\/C. gattii species complex. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0037566"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3669","DOI":"10.1128\/JCM.01072-09","article-title":"Identification of Cryptococcus gattii by use of L-canavanine glycine bromothymol blue medium and DNA sequencing","volume":"47","author":"Klein","year":"2009","journal-title":"J. Clin. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"675213","DOI":"10.1155\/2013\/675213","article-title":"Global Molecular Epidemiology of Cryptococcus neoformans and Cryptococcus gattii: An Atlas of the Molecular Types","volume":"2013","author":"Cogliati","year":"2013","journal-title":"Scientifica"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"S109","DOI":"10.1093\/cid\/ciaa1836","article-title":"Molecular Techniques for Genus and Species Determination of Fungi From Fresh and Paraffin-Embedded Formalin-Fixed Tissue in the Revised EORTC\/MSGERC Definitions of Invasive Fungal Infection","volume":"72","author":"Lockhart","year":"2021","journal-title":"Clin. Infect. Dis."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1016\/S1567-1356(03)00176-4","article-title":"The internal transcribed spacers and 5.8S rRNA gene show extensive diversity among isolates of the Cryptococcus neoformans species complex","volume":"4","author":"Katsu","year":"2004","journal-title":"FEMS Yeast Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1383","DOI":"10.1099\/jmm.0.000575","article-title":"Detection of fungal pathogens by a new broad range real-time PCR assay targeting the fungal ITS2 region","volume":"66","author":"Zeller","year":"2017","journal-title":"J. Med. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1093\/mmy\/myv092","article-title":"Clinical utility of panfungal polymerase chain reaction for the diagnosis of invasive fungal disease: A single center experience","volume":"54","author":"Trubiano","year":"2016","journal-title":"Med. Mycol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1177\/0897190017737697","article-title":"Cost Justification of the BioFire FilmArray Meningitis\/Encephalitis Panel Versus Standard of Care for Diagnosing Meningitis in a Community Hospital","volume":"32","author":"Soucek","year":"2019","journal-title":"J. Pharm. Pract."}],"container-title":["Journal of Fungi"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2309-608X\/8\/5\/462\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:04:14Z","timestamp":1760137454000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2309-608X\/8\/5\/462"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,4,29]]},"references-count":31,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2022,5]]}},"alternative-id":["jof8050462"],"URL":"https:\/\/doi.org\/10.3390\/jof8050462","relation":{},"ISSN":["2309-608X"],"issn-type":[{"value":"2309-608X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,4,29]]}}}