Pinheiro Breno Gonçalves, Pôssa Ana Paula, Della Terra Paula Portella, de Carvalho Jamile Ambrósio, Ricci Giannina, Nishikaku Angela Satie, Hahn Rosane Christine, Camargo Zoilo Pires de, Rodrigues Anderson Messias
Laboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of São Paulo (UNIFESP), São Paulo 04023062, Brazil.
Department of Medicine, Discipline of infectious Diseases, Federal University of São Paulo (UNIFESP), São Paulo 04023062, Brazil.
J Fungi (Basel). 2021 Feb 26;7(3):169. doi: 10.3390/jof7030169.
Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the complex and . 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 complex and 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 species. Primers PbraCx-F and PbraCx-R targeting DNA produced an amplicon of 308 bp, while primers Plu-F and Plu-R targeting 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 revealed 100% specificity (AUC = 1.000, 95%CI 0.972-1.000, < 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 complex ( = 7) or ( = 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 (S1) and in the same biopsy. Our duplex PCR assay is useful to detect and differentiate members of the complex and , 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.
副球孢子菌病（PCM）是一种由 复合体和 的成员引起的危及生命的系统性真菌感染。由于表型重叠，使用经典真菌学方法对PCM进行常规诊断直至物种水平并不特异。迫切需要特异性、灵敏且经济高效的分子工具来诊断PCM。利用gp43基因外显子2的变异设计物种特异性引物对，以在双重PCR检测中区分 复合体和 的成员。引物-BLAST搜索揭示了高度物种特异性的引物，除了 物种外，未发现与DNA数据库有显著同源区域。靶向 DNA的引物PbraCx-F和PbraCx-R产生了308 bp 的扩增子，而靶向 DNA的引物Plu-F和Plu-R产生了142 bp 的扩增子。我们的双重PCR检测的检测下限为1 pg基因组DNA（gDNA）。一组62份 显示出100%的特异性（AUC = 1.000，95%CI 0.972 - 1.000， < 0.0001），且不与其他医学相关真菌或人类DNA发生交叉反应。作为概念验证，我们展示了从PCM患者器官的广泛福尔马林固定、石蜡包埋（FFPE）组织中准确鉴定 复合体（ = 7）或 （ = 6）。在4例病例中，FFPE PCR结果首次证实了同一活检组织中存在 （S1）和 的共同感染。我们的双重PCR检测可用于检测和区分 复合体和 的成员，为临床实验室提供了一种重要的常规应用工具，特别是在非典型病例中，如血清学阴性和临床标本真菌学检查阳性的病例以及疑似共同感染病例的调查中。这可能每年使美洲广大地区的数千名感染患者受益。
