A New Duplex PCR-Assay for the Detection and Identification of Paracoccidioides Species

Author(s)

Pinheiro, Breno Goncalves ^[1] ; Possa, Ana Paula ^{[2, 1]} ; Della Terra, Paula Portella ^{[2, 1]} ; de Carvalho, Jamile Ambrosio ^[1] ; Ricci, Giannina ^[3] ; Nishikaku, Angela Satie ^[3] ; Hahn, Rosane Christine ^{[4, 5]} ; Camargo, Zoilo Pires de ^{[2, 1]} ; Rodrigues, Anderson Messias ^{[2, 1]}

Affiliation

^[1] Fed Univ Sao Paulo UNIFESP, Lab Emerging Fungal Pathogens, Dept Microbiol Immunol & Parasitol, Discipline Cellular Biol, BR-04023062 Sao Paulo - Brazil

^[2] Fed Univ Sao Paulo UNIFESP, Dept Med, Discipline Infect Dis, BR-04023062 Sao Paulo - Brazil

^[3] Ctr Diagnost & Pesquisa Biol Mol Dr Ivo Ricci, BR-13561020 Sao Paulo - Brazil

^[4] Univ Fed Mato Grosso, Julio Muller Univ Hosp, BR-78048902 Cuiaba - Brazil

^[5] Univ Fed Mato Grosso, Lab Mycol Res, Fac Med, BR-78060900 Cuiaba - Brazil

• Document type: Journal article
• Source: JOURNAL OF FUNGI; v. 7, n. 3 MAR 2021.
• Web of Science Citations: 0

Abstract

Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the Paracoccidioides brasiliensis complex and P. lutzii. 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 P. brasiliensis complex and P. lutzii 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 Paracoccidioides species. Primers PbraCx-F and PbraCx-R targeting P. brasiliensis DNA produced an amplicon of 308 bp, while primers Plu-F and Plu-R targeting P. lutzii 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 Paracoccidioides revealed 100% specificity (AUC = 1.000, 95%CI 0.972-1.000, p < 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 P. brasiliensis complex (n = 7) or P. lutzii (n = 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 P. brasiliensis (S1) and P. lutzii in the same biopsy. Our duplex PCR assay is useful to detect and differentiate members of the P. brasiliensis complex and P. lutzii, 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. (AU)

• FAPESP's process: 17/27265-5 - Molecular epidemiology and genomic perspectives on the evolution and spread of emerging fungal pathogens
• Grantee: Anderson Messias Rodrigues
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 18/21460-3 - Study of different antigenic preparations of Paracoccidioides lutzii for the standardization of the ELISA test as an aid in the diagnosis of paracoccidioidomycosis due to P. lutzii
• Grantee: Zoilo Pires de Camargo
• Support Opportunities: Regular Research Grants