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Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents

Classic paracoccidioidomycosis (PCM) is a potentially deadly neglected tropical systemic mycosis caused by members of the Paracoccidioides brasiliensis complex (P. brasiliensis s. str., P. americana, P. restrepiensis, and P. venezuelensis) and P. lutzii. The laboratorial diagnosis of PCM relies on o...

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Autores principales: Pinheiro, Breno Gonçalves, Pôssa, Ana Paula, Ricci, Giannina, Nishikaku, Angela Satie, Hagen, Ferry, Hahn, Rosane Christine, de Camargo, Zoilo Pires, Rodrigues, Anderson Messias
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10057483/
https://www.ncbi.nlm.nih.gov/pubmed/36983526
http://dx.doi.org/10.3390/jof9030358
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author Pinheiro, Breno Gonçalves
Pôssa, Ana Paula
Ricci, Giannina
Nishikaku, Angela Satie
Hagen, Ferry
Hahn, Rosane Christine
de Camargo, Zoilo Pires
Rodrigues, Anderson Messias
author_facet Pinheiro, Breno Gonçalves
Pôssa, Ana Paula
Ricci, Giannina
Nishikaku, Angela Satie
Hagen, Ferry
Hahn, Rosane Christine
de Camargo, Zoilo Pires
Rodrigues, Anderson Messias
author_sort Pinheiro, Breno Gonçalves
collection PubMed
description Classic paracoccidioidomycosis (PCM) is a potentially deadly neglected tropical systemic mycosis caused by members of the Paracoccidioides brasiliensis complex (P. brasiliensis s. str., P. americana, P. restrepiensis, and P. venezuelensis) and P. lutzii. The laboratorial diagnosis of PCM relies on observing pathognomonic structures such as the “steering wheel” or “Mickey Mouse” 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 0.964–1.000, p < 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 ± 0.05, 95% CI = 0.756–0.979, p < 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 0.872–1.000, p < 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.
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spelling pubmed-100574832023-03-30 Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents Pinheiro, Breno Gonçalves Pôssa, Ana Paula Ricci, Giannina Nishikaku, Angela Satie Hagen, Ferry Hahn, Rosane Christine de Camargo, Zoilo Pires Rodrigues, Anderson Messias J Fungi (Basel) Article Classic paracoccidioidomycosis (PCM) is a potentially deadly neglected tropical systemic mycosis caused by members of the Paracoccidioides brasiliensis complex (P. brasiliensis s. str., P. americana, P. restrepiensis, and P. venezuelensis) and P. lutzii. The laboratorial diagnosis of PCM relies on observing pathognomonic structures such as the “steering wheel” or “Mickey Mouse” 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 0.964–1.000, p < 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 ± 0.05, 95% CI = 0.756–0.979, p < 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 0.872–1.000, p < 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. MDPI 2023-03-15 /pmc/articles/PMC10057483/ /pubmed/36983526 http://dx.doi.org/10.3390/jof9030358 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Pinheiro, Breno Gonçalves
Pôssa, Ana Paula
Ricci, Giannina
Nishikaku, Angela Satie
Hagen, Ferry
Hahn, Rosane Christine
de Camargo, Zoilo Pires
Rodrigues, Anderson Messias
Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents
title Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents
title_full Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents
title_fullStr Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents
title_full_unstemmed Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents
title_short Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents
title_sort development of a multiplex qpcr assay for fast detection and differentiation of paracoccidioidomycosis agents
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10057483/
https://www.ncbi.nlm.nih.gov/pubmed/36983526
http://dx.doi.org/10.3390/jof9030358
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