A New Duplex PCR-Assay for the Detection and Identification of <i>Paracoccidioides</i> Species
Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the <i>Paracoccidioides brasiliensis</i> complex and <i>P. lutzii</i>. Routine diagnoses of PCM down to the species level using classical mycological approaches are unspecific du...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-02-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/7/3/169 |
| _version_ | 1797395200363986944 |
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| author | Breno Gonçalves Pinheiro Ana Paula Pôssa Paula Portella Della Terra Jamile Ambrósio de Carvalho Giannina Ricci Angela Satie Nishikaku Rosane Christine Hahn Zoilo Pires de Camargo Anderson Messias Rodrigues |
| author_facet | Breno Gonçalves Pinheiro Ana Paula Pôssa Paula Portella Della Terra Jamile Ambrósio de Carvalho Giannina Ricci Angela Satie Nishikaku Rosane Christine Hahn Zoilo Pires de Camargo Anderson Messias Rodrigues |
| author_sort | Breno Gonçalves Pinheiro |
| collection | DOAJ |
| description | Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the <i>Paracoccidioides brasiliensis</i> complex and <i>P. lutzii</i>. Routine diagnoses of PCM down to the species level using classical mycological approaches are unspecific due to overlapping phenotypes. There is an urgent need for specific, sensitive, and cost-effective molecular tools to diagnose PCM. Variation among the exon-2 of the gp43 gene was exploited to design species-specific primer pairs to discriminate between members of the <i>P. brasiliensis</i> complex and <i>P. lutzii</i> in a duplex PCR assay. Primer-BLAST searches revealed highly species-specific primers, and no significant region of homology was found against DNA databases except for <i>Paracoccidioides</i> species. Primers PbraCx-F and PbraCx-R targeting <i>P. brasiliensis</i> DNA produced an amplicon of 308 bp, while primers Plu-F and Plu-R targeting <i>P. lutzii</i> DNA generated an amplicon of 142 bp. The lower limit of detection for our duplex PCR assay was 1 pg of gDNA. A panel of 62 <i>Paracoccidioides</i> revealed 100% specificity (AUC = 1.000, 95%CI 0.972–1.000, <i>p</i> < 0.0001) without cross-reacting with other medically relevant fungi or human DNA. As a proof of concept, we demonstrated the accurate identification of the <i>P. brasiliensis</i> complex (<i>n</i> = 7) or <i>P. lutzii</i> (<i>n</i> = 6) from a broad range of formalin-fixed, paraffin-embedded (FFPE) tissues of PCM patient’s organs. In four cases, FFPE PCR results confirmed, for the first time, co-infection due to <i>P. brasiliensis</i> (S1) and <i>P. lutzii</i> in the same biopsy. Our duplex PCR assay is useful to detect and differentiate members of the <i>P. brasiliensis</i> complex and <i>P. lutzii</i>, providing clinical laboratories with an important tool to be applied routinely, especially in atypical cases such as those featuring negative serology and positive mycological examination of clinical specimens as well as for the investigation of putative co-infection cases. This will likely benefit thousands of infected patients every year in a wide area of the Americas. |
| first_indexed | 2024-03-09T00:31:02Z |
| format | Article |
| id | doaj.art-ef9a2014654941aaa70f5a6d8a6be245 |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-09T00:31:02Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-ef9a2014654941aaa70f5a6d8a6be2452023-12-11T18:32:02ZengMDPI AGJournal of Fungi2309-608X2021-02-017316910.3390/jof7030169A New Duplex PCR-Assay for the Detection and Identification of <i>Paracoccidioides</i> SpeciesBreno Gonçalves Pinheiro0Ana Paula Pôssa1Paula Portella Della Terra2Jamile Ambrósio de Carvalho3Giannina Ricci4Angela Satie Nishikaku5Rosane Christine Hahn6Zoilo Pires de Camargo7Anderson Messias Rodrigues8Laboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of São Paulo (UNIFESP), São Paulo 04023062, BrazilLaboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of São Paulo (UNIFESP), São Paulo 04023062, BrazilLaboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of São Paulo (UNIFESP), São Paulo 04023062, BrazilLaboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of São Paulo (UNIFESP), São Paulo 04023062, BrazilCentro de Diagnóstico e Pesquisa em Biologia Molecular Dr. Ivo Ricci, São Paulo 13561020, BrazilCentro de Diagnóstico e Pesquisa em Biologia Molecular Dr. Ivo Ricci, São Paulo 13561020, BrazilLaboratory of Mycology/Research, Faculty of Medicine, Federal University of Mato Grosso, Cuiabá 78060900, BrazilLaboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of São Paulo (UNIFESP), São Paulo 04023062, BrazilLaboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of São Paulo (UNIFESP), São Paulo 04023062, BrazilParacoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the <i>Paracoccidioides brasiliensis</i> complex and <i>P. lutzii</i>. Routine diagnoses of PCM down to the species level using classical mycological approaches are unspecific due to overlapping phenotypes. There is an urgent need for specific, sensitive, and cost-effective molecular tools to diagnose PCM. Variation among the exon-2 of the gp43 gene was exploited to design species-specific primer pairs to discriminate between members of the <i>P. brasiliensis</i> complex and <i>P. lutzii</i> in a duplex PCR assay. Primer-BLAST searches revealed highly species-specific primers, and no significant region of homology was found against DNA databases except for <i>Paracoccidioides</i> species. Primers PbraCx-F and PbraCx-R targeting <i>P. brasiliensis</i> DNA produced an amplicon of 308 bp, while primers Plu-F and Plu-R targeting <i>P. lutzii</i> DNA generated an amplicon of 142 bp. The lower limit of detection for our duplex PCR assay was 1 pg of gDNA. A panel of 62 <i>Paracoccidioides</i> revealed 100% specificity (AUC = 1.000, 95%CI 0.972–1.000, <i>p</i> < 0.0001) without cross-reacting with other medically relevant fungi or human DNA. As a proof of concept, we demonstrated the accurate identification of the <i>P. brasiliensis</i> complex (<i>n</i> = 7) or <i>P. lutzii</i> (<i>n</i> = 6) from a broad range of formalin-fixed, paraffin-embedded (FFPE) tissues of PCM patient’s organs. In four cases, FFPE PCR results confirmed, for the first time, co-infection due to <i>P. brasiliensis</i> (S1) and <i>P. lutzii</i> in the same biopsy. Our duplex PCR assay is useful to detect and differentiate members of the <i>P. brasiliensis</i> complex and <i>P. lutzii</i>, providing clinical laboratories with an important tool to be applied routinely, especially in atypical cases such as those featuring negative serology and positive mycological examination of clinical specimens as well as for the investigation of putative co-infection cases. This will likely benefit thousands of infected patients every year in a wide area of the Americas.https://www.mdpi.com/2309-608X/7/3/169molecular diagnosticsspecies-specific PCRduplex PCR<i>Paracoccidioides brasiliensis</i><i>Paracoccidioides lutzii</i>paracoccidioidomycosis |
| spellingShingle | Breno Gonçalves Pinheiro Ana Paula Pôssa Paula Portella Della Terra Jamile Ambrósio de Carvalho Giannina Ricci Angela Satie Nishikaku Rosane Christine Hahn Zoilo Pires de Camargo Anderson Messias Rodrigues A New Duplex PCR-Assay for the Detection and Identification of <i>Paracoccidioides</i> Species Journal of Fungi molecular diagnostics species-specific PCR duplex PCR <i>Paracoccidioides brasiliensis</i> <i>Paracoccidioides lutzii</i> paracoccidioidomycosis |
| title | A New Duplex PCR-Assay for the Detection and Identification of <i>Paracoccidioides</i> Species |
| title_full | A New Duplex PCR-Assay for the Detection and Identification of <i>Paracoccidioides</i> Species |
| title_fullStr | A New Duplex PCR-Assay for the Detection and Identification of <i>Paracoccidioides</i> Species |
| title_full_unstemmed | A New Duplex PCR-Assay for the Detection and Identification of <i>Paracoccidioides</i> Species |
| title_short | A New Duplex PCR-Assay for the Detection and Identification of <i>Paracoccidioides</i> Species |
| title_sort | new duplex pcr assay for the detection and identification of i paracoccidioides i species |
| topic | molecular diagnostics species-specific PCR duplex PCR <i>Paracoccidioides brasiliensis</i> <i>Paracoccidioides lutzii</i> paracoccidioidomycosis |
| url | https://www.mdpi.com/2309-608X/7/3/169 |
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