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The laboratorial diagnosis of PCM relies on observing pathognomonic structures such as the \u201csteering wheel\u201d or \u201cMickey Mouse\u201d shape in the direct mycological examination, fresh biopsied tissue in 10% KOH, histopathological analysis, and\/or the isolation of the fungus in culture. However, these procedures are time-consuming and do not allow for the speciation of Paracoccidioides due to overlapping morphologies. Here, we propose a new one-tube multiplex probe-based qPCR assay to detect and recognize agents of the P. brasiliensis complex and P. lutzii. Primers (Paracoco-F and Paracoco-R) and TaqMan probes (PbraCx-Fam, Plu-Ned, and Paracoco-Vic) were developed to target the rDNA (ITS2\/28S) in the Paracoccidioides genome. A panel of 77 Paracoccidioides isolates revealed a 100% specificity (AUC = 1.0, 95% CI 0.964\u20131.000, p &lt; 0.0001) without cross-reacting with other medically relevant fungi or human and murine DNA. The lower limit of detection was 10 fg of gDNA and three copies of the partial rDNA amplicon. Speciation using qPCR was in perfect agreement with AFLP and TUB1-RFLP markers (kappa = 1.0). As a proof of concept, we assessed a panel of 16 formalin-fixed and paraffin-embedded specimens from histopathologically confirmed PCM patients to reveal a significant sensitivity of 81.25% and specificity of 100% (AUC = 0.906 \u00b1 0.05, 95% CI = 0.756\u20130.979, p &lt; 0.0001, Youden index J = 0.8125). Our assay achieved maximum sensitivity (100%) and specificity (100%) using fresh clinical samples (n = 9) such as sputum, bronchoalveolar lavage, and tissue fragments from PCM patients (AUC = 1.0, 95% CI 0.872\u20131.000, p &lt; 0.0001, Youden index J = 1.0). Overall, our qPCR assay simplifies the molecular diagnosis of PCM and can be easily implemented in any routine laboratory, decreasing a critical bottleneck for the early treatment of PCM patients across a vast area of the Americas.<\/jats:p>","DOI":"10.3390\/jof9030358","type":"journal-article","created":{"date-parts":[[2023,3,15]],"date-time":"2023-03-15T05:22:59Z","timestamp":1678857779000},"page":"358","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9134-0657","authenticated-orcid":false,"given":"Breno Gon\u00e7alves","family":"Pinheiro","sequence":"first","affiliation":[{"name":"Laboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of S\u00e3o Paulo (UNIFESP), S\u00e3o Paulo 04023-062, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana Paula","family":"P\u00f4ssa","sequence":"additional","affiliation":[{"name":"Laboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of S\u00e3o Paulo (UNIFESP), S\u00e3o Paulo 04023-062, Brazil"},{"name":"Department of Medicine, Discipline of Infectious Diseases, Federal University of S\u00e3o Paulo (UNIFESP), S\u00e3o Paulo 04023-062, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giannina","family":"Ricci","sequence":"additional","affiliation":[{"name":"Centro de Diagn\u00f3stico e Pesquisa em Biologia Molecular Dr. Ivo Ricci, S\u00e3o Carlos 13561-020, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9279-0844","authenticated-orcid":false,"given":"Angela Satie","family":"Nishikaku","sequence":"additional","affiliation":[{"name":"Centro de Diagn\u00f3stico e Pesquisa em Biologia Molecular Dr. Ivo Ricci, S\u00e3o Carlos 13561-020, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5622-1916","authenticated-orcid":false,"given":"Ferry","family":"Hagen","sequence":"additional","affiliation":[{"name":"Department of Medical Mycology, Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands"},{"name":"Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam, Sciencepark 904, 1098 XH Amsterdam, The Netherlands"},{"name":"Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rosane Christine","family":"Hahn","sequence":"additional","affiliation":[{"name":"Laboratory of Mycology\/Research, Faculty of Medicine, Federal University of Mato Grosso, Cuiab\u00e1 78060-900, Brazil"},{"name":"J\u00falio Muller University Hospital, Federal University of Mato Grosso, Cuiab\u00e1 78048-902, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zoilo Pires","family":"de Camargo","sequence":"additional","affiliation":[{"name":"Laboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of S\u00e3o Paulo (UNIFESP), S\u00e3o Paulo 04023-062, Brazil"},{"name":"Department of Medicine, Discipline of Infectious Diseases, Federal University of S\u00e3o Paulo (UNIFESP), S\u00e3o Paulo 04023-062, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0891-4563","authenticated-orcid":false,"given":"Anderson Messias","family":"Rodrigues","sequence":"additional","affiliation":[{"name":"Laboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of S\u00e3o Paulo (UNIFESP), S\u00e3o Paulo 04023-062, Brazil"},{"name":"Department of Medicine, Discipline of Infectious Diseases, Federal University of S\u00e3o Paulo (UNIFESP), S\u00e3o Paulo 04023-062, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,3,15]]},"reference":[{"key":"ref_1","unstructured":"Rodrigues, A.M., Hagen, F., Puccia, R., Hahn, R.C., and de Camargo, Z.P. 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