新型荚膜组织胞浆菌定量 RT-PCR 检测方法的评估。
Evaluation of a New Histoplasma spp. Quantitative RT-PCR Assay.
机构信息
Laboratoire de Parasitologie-Mycologie, Groupe Hospitalier Saint-Louis-Lariboisière-Fernand-Widal, Assistance Publique-Hôpitaux de Paris, Paris, France; Molecular Mycology Unit, CNRS UMR2000, Institut Pasteur, Paris, France; National Reference Center for Invasive Mycoses and Antifungals, Institut Pasteur, Paris, France; UFR de Médecine, Hôpital Necker Enfants malades, Assistance Publique-Hôpitaux de Paris, IHU Imagine, Paris Descartes University, Université de Paris, Paris, France.
Laboratoire de Parasitologie-Mycologie, Groupe Hospitalier Saint-Louis-Lariboisière-Fernand-Widal, Assistance Publique-Hôpitaux de Paris, Paris, France; Molecular Mycology Unit, CNRS UMR2000, Institut Pasteur, Paris, France; UFR de Médecine, Hôpital Necker Enfants malades, Assistance Publique-Hôpitaux de Paris, IHU Imagine, Paris Descartes University, Université de Paris, Paris, France.
出版信息
J Mol Diagn. 2021 Jun;23(6):698-709. doi: 10.1016/j.jmoldx.2021.02.007. Epub 2021 Mar 9.
Laboratory diagnosis of histoplasmosis is based on various methods, including microscopy, culture, antigen, and DNA detection of Histoplasma capsulatum var. capsulatum or Histoplasma capsulatum var. duboisii. To improve sensitivity of existing real-time quantitative PCR (qPCR) assays, we developed a new RT-qPCR assay that allows amplification of whole nucleic acids of Histoplasma spp. validated on suspected cases. The limit of detection was 20 copies, and the specificity against 114 fungal isolates/species was restricted to Histoplasma spp. Whole nucleic acids of 1319 prospectively collected consecutive samples from 907 patients suspected of having histoplasmosis were tested routinely between May 2015 and May 2019 in parallel with standard diagnostic procedures performed in parallel. Forty-four had proven histoplasmosis attributable to H. capsulatum var. capsulatum (n = 40) or H. capsulatum var. duboisii (n = 4) infections. The results of RT-qPCR were positive in 43 of 44 patients (97.7% sensitivity) in at least one specimen. Nine of 863 cases (99% specificity) were RT-qPCR positive and therefore classified as possible cases. RT-qPCR was positive in 13 of 30 (43.3%) blood samples tested in proven cases. A positive RT-qPCR result in blood was significantly associated with H. capsulatum var. capsulatum progressively disseminated histoplasmosis with a positive RT-qPCR result in 92.3% of the immunocompromised patients with disseminated disease. This new Histoplasma RT-qPCR assay enabling amplification of H. capsulatum var. capsulatum and H. capsulatum var. duboisii is highly sensitive and allows the diagnosis of histoplasmosis advantageously from blood and bronchoalveolar lavage fluid.
组织胞浆菌病的实验室诊断基于各种方法，包括显微镜检查、培养、抗原和 Histoplasma capsulatum var. capsulatum 或 Histoplasma capsulatum var. duboisii 的 DNA 检测。为了提高现有实时定量 PCR（qPCR）检测的灵敏度，我们开发了一种新的 RT-qPCR 检测方法，该方法允许扩增 Histoplasma spp. 的整个核酸，在疑似病例中进行了验证。检测限为 20 个拷贝，对 114 种真菌分离株/种的特异性仅限于 Histoplasma spp. 在 2015 年 5 月至 2019 年 5 月期间，对 907 例疑似患有组织胞浆菌病的患者连续采集的 1319 例前瞻性连续样本，与并行进行的标准诊断程序平行进行常规检测。44 例有明确的组织胞浆菌病归因于 H. capsulatum var. capsulatum（n=40）或 H. capsulatum var. duboisii（n=4）感染。在至少一个标本中，RT-qPCR 结果在 44 例患者中的 43 例（97.7%的灵敏度）中为阳性。在 863 例病例中，有 9 例（99%的特异性）RT-qPCR 阳性，因此被归类为可能病例。在确诊病例中，有 13 例（43.3%）血液样本 RT-qPCR 阳性。血液中 RT-qPCR 阳性结果与 H. capsulatum var. capsulatum 进行性播散性组织胞浆菌病显著相关，在播散性疾病的免疫功能低下患者中，RT-qPCR 阳性结果的比例为 92.3%。这种新的 Histoplasma RT-qPCR 检测方法能够扩增 H. capsulatum var. capsulatum 和 H. capsulatum var. duboisii，具有高度的灵敏度，有利于从血液和支气管肺泡灌洗液中诊断组织胞浆菌病。
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