用于快速检测和区分副球孢子菌病病原体的多重定量聚合酶链反应检测方法的开发

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

作者信息

Pinheiro Breno Gonçalves, Pôssa Ana Paula, Ricci Giannina, Nishikaku Angela Satie, Hagen Ferry, Hahn Rosane Christine, de Camargo Zoilo Pires, Rodrigues Anderson Messias

机构信息

Laboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of São Paulo (UNIFESP), São Paulo 04023-062, Brazil.

Department of Medicine, Discipline of Infectious Diseases, Federal University of São Paulo (UNIFESP), São Paulo 04023-062, Brazil.

出版信息

J Fungi (Basel). 2023 Mar 15;9(3):358. doi: 10.3390/jof9030358.

Classic paracoccidioidomycosis (PCM) is a potentially deadly neglected tropical systemic mycosis caused by members of the complex (, , , and ) and . 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 due to overlapping morphologies. Here, we propose a new one-tube multiplex probe-based qPCR assay to detect and recognize agents of the complex and . 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 genome. A panel of 77 isolates revealed a 100% specificity (AUC = 1.0, 95% CI 0.964-1.000, < 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 -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, < 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, < 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.

经典副球孢子菌病（PCM）是一种由副球孢子菌复合体（副球孢子菌 brasiliensis、副球孢子菌 lutzii、副球孢子菌 americana 和副球孢子菌 venezuelensis）及其他菌种引起的潜在致命性被忽视的热带系统性真菌病。PCM 的实验室诊断依赖于在直接真菌学检查、10%氢氧化钾处理的新鲜活检组织、组织病理学分析和/或培养中分离真菌时观察到特征性结构，如“方向盘”或“米老鼠”形状。然而，这些程序耗时且由于形态重叠无法对副球孢子菌进行菌种鉴定。在此，我们提出一种基于单管多重探针的新型 qPCR 检测方法，用于检测和识别副球孢子菌复合体及其他菌种。开发了引物（Paracoco-F 和 Paracoco-R）和 TaqMan 探针（PbraCx-Fam、Plu-Ned 和 Paracoco-Vic），以靶向副球孢子菌基因组中的 rDNA（ITS2/28S）。一组 77 株副球孢子菌分离株显示出 100%的特异性（AUC = 1.0，95%CI 0.964 - 1.000，P < 0.0001），且不与其他医学相关真菌或人和小鼠 DNA 发生交叉反应。检测下限为 10 fg 的基因组 DNA 和三个拷贝的部分 rDNA 扩增子片段。使用 qPCR 进行的菌种鉴定与 AFLP 和 rDNA-RFLP 标记完全一致（kappa = 1.0）。作为概念验证，我们评估了一组来自组织病理学确诊的 PCM 患者的 16 份福尔马林固定石蜡包埋标本，显示出显著的敏感性为 81.25%，特异性为 100%（AUC = 0.906 ± 0.05，95%CI = 0.756 - 0.979，P < 0.0001，约登指数 J = 0.8125）。使用来自 PCM 患者的新鲜临床样本（n = 9），如痰液、支气管肺泡灌洗和组织碎片，我们的检测方法实现了最大敏感性（100%）和特异性（100%）（AUC = 1.0，95%CI 0.872 - 1.000，P < 0.0001，约登指数 J = 1.0）。总体而言，我们的 qPCR 检测方法简化了 PCM 的分子诊断，可在任何常规实验室轻松实施，减少了美洲广大地区 PCM 患者早期治疗中的关键瓶颈。